Thienopyrrole compounds and uses thereof

ABSTRACT

Thienopyrrole compounds that may inhibit Oplophorus-derived luciferases are disclosed, as well as compositions and kits comprising the thienopyrrole compounds, and methods of using the thienopyrrole compounds.

CROSS-REFERENCE To RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/184,714, filed Jun. 25, 2015, and U.S. Provisional Application No.62/206,525, filed on Aug. 18, 2015, the entire contents of each of whichis incorporated herein by reference.

TECHNICAL FIELD

The present disclosure is directed to thienopyrrole compounds that mayinhibit Oplophorus-derived luciferases.

BACKGROUND

Reporter molecules are routinely used to monitor molecular events in thefields of biology, biochemistry, immunology, cell biology, and molecularbiology. Luciferases based on the luciferase secreted from the deep-seashrimp, Oplophorus gracilirostris, may be used as reporter molecules andhave been shown to have advantageous characteristics including broadsubstrate specificity, high activity, and high quantum yield. It may befurther advantageous, in certain applications, to control theluminescent signal from Oplophorus luciferases.

SUMMARY

In one aspect, the disclosure provides a compound of formula (I), or asalt thereof:

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl; and

R³ and R⁴ are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring.

In one aspect, the disclosure provides a method of inhibiting anOplophorus-derived luciferase the method comprising contacting theOplophorus-derived luciferase with a compound described herein, such asa compound of formula (I), (Ia), (Ib), (Ib′) or (II).

In one aspect, the disclosure provides a method of inhibiting anOplophorus-derived luciferase, the method comprising contacting theOplophorus-derived luciferase with a compound of formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring.

In one aspect, the disclosure provides a method for modulatingluminescence of an Oplophorus-derived luciferase in a sample, the methodcomprising,

(a) contacting the sample with a coelenterazine substrate and a compounddescribed herein, such as a compound of formula (I), (Ia), (Ib), (Ib′)or (II); and

(b) detecting luminescence in the sample,

wherein the compound causes a decrease in the luminescence from theOplophorus-derived luciferase.

In one aspect, the disclosure provides a method to detect an interactionbetween a first protein and a second protein in a sample, the methodcomprising:

(a) contacting a sample with a coelenterazine substrate a compounddescribed herein, such as a compound of formula (I), (Ia), (Ib), (Ib′)or (II), wherein the sample comprises:

-   -   (i) a first polynucleotide encoding a first fusion protein,        wherein the first fusion protein comprises a first fragment of        an Oplophorus-derived luciferase and a first protein; and    -   (ii) a second polynucleotide encoding a second fusion protein,        wherein the second fusion protein comprises a second fragment of        the Oplophorus-derived luciferase and a second protein; and

(b) detecting luminescence in the sample,

wherein the detection of luminescence indicates an interaction betweenthe first protein and the second protein.

In one aspect, the disclosure provides a method to detect an interactionbetween a first protein and a second protein in a sample, the methodcomprising:

(a) contacting a sample with a coelenterazine substrate and a compounddescribed herein, such as a compound of formula (I), (Ia), (Ib), (Ib′)or (II), wherein the sample comprises:

-   -   (iii) a first polynucleotide encoding a first fusion protein,        wherein the first fusion protein comprises an Oplophorus-derived        luciferase and a first protein, wherein the Oplophorus-derived        luciferase is a bioluminescent donor; and    -   (iv) a second polynucleotide encoding a second fusion protein,        wherein the second fusion protein comprises a fluorescent        acceptor molecule and a second protein;

(b) detecting bioluminescence resonance energy transfer (BRET) in thesample, indicating an interaction or close proximity of thebioluminescent donor and the fluorescence acceptor.

In one aspect, the disclosure provides a bioluminescence resonanceenergy transfer (BRET) system comprising: a first fusion proteinincluding a first target protein and a bioluminescence donor molecule,wherein the bioluminescence donor molecule is an Oplophorus-derivedluciferase; a second fusion protein including a second target proteinand a fluorescent acceptor molecule; a coelenterazine substrate, and acompound described herein, such as a compound of formula (I), (Ia),(Ib), (Ib′) or (II).

In one aspect, the disclosure provides a kit comprising:

(a) a compound described herein, such as a compound of formula (I),(Ia), (Ib), (Ib′) or (II); and

(b) an Oplophorus-derived luciferase.

In one aspect, the disclosure provides a method for modulatingluminescence of an Oplophorus-derived luciferase in a sample, the methodcomprising:

(a) contacting the sample with a coelenterazine substrate and a compoundof formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring; and

(b) detecting luminescence in the sample,

wherein the compound of formula (II) causes a decrease in theluminescence from the Oplophorus-derived luciferase.

In one aspect, the disclosure provides a method to detect an interactionbetween a first protein and a second protein in a sample, the methodcomprising:

(a) contacting a sample with a coelenterazine substrate and a compoundof formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring,

wherein the sample comprises:

-   -   (v) a first polynucleotide encoding a first fusion protein,        wherein the first fusion protein comprises a first fragment of        an Oplophorus-derived luciferase and a first protein; and    -   (vi) a second polynucleotide encoding a second fusion protein,        wherein the second fusion protein comprises a second fragment of        the Oplophorus-derived luciferase and a second protein; and

(b) detecting luminescence in the sample,

wherein the detection of luminescence indicates an interaction betweenthe first protein and the second protein.

In one aspect, the disclosure provides a method to detect an interactionbetween a first protein and a second protein in a sample, the methodcomprising:

(a) contacting a sample with a coelenterazine substrate and a compoundof formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring,

wherein the sample comprises:

-   -   (vii) a first polynucleotide encoding a first fusion protein,        wherein the first fusion protein comprises an Oplophorus-derived        luciferase and a first protein, wherein the Oplophorus-derived        luciferase is a bioluminescent donor; and    -   (viii) a second polynucleotide encoding a second fusion protein,        wherein the second fusion protein comprises a fluorescent        acceptor molecule and a second protein;

(b) detecting bioluminescence resonance energy transfer (BRET) in thesample, indicating an interaction or close proximity of thebioluminescent donor and the fluorescence acceptor.

In one aspect, the disclosure provides a bioluminescence resonanceenergy transfer (BRET) system comprising: a first fusion proteinincluding a first target protein and a bioluminescence donor molecule,wherein the bioluminescence donor molecule is an Oplophorus-derivedluciferase; a second fusion protein including a second target proteinand a fluorescent acceptor molecule; a coelenterazine substrate, and acompound of formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring.

In one aspect, the disclosure provides a kit comprising:

(a) a compound of formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring; and

(b) an Oplophorus-derived luciferase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibition of NANOLUC® (Nluc) enzyme by JRW-0004 in amultiplex assay that combines RealTime-Glo and CASPASE-GLO® assaysystem.

FIG. 2 shows the inhibition of Nluc by thienopyrrole compounds JRW-0004,JRW-0013, JRW-0006, JRW-0042, JRW-0138, and JRW-0147.

FIG. 3 shows the thienopyrrole compounds JRW-0004, JRW-0013, JRW-0006,JRW-0042, JRW-0138, and JRW-0147 do not inhibit firefly luciferaseactivity.

FIGS. 4A-4D show that thienopyrrole compounds JRW-0004 (FIGS. 4A and 4B)and WZ141-88 (FIGS. 4C and 4D) inhibit Nluc in a dose- andtime-dependent manner.

FIGS. 5A-5D show that thienopyrrole compounds WZ141-86 (FIGS. 5A and5B), WZ141-74 (FIG. 5C), and WZ141-84 (FIG. 5D) inhibit Nluc in a dose-and time-dependent manner.

FIGS. 6A-6C show that thienopyrrole compounds WZ141-89 (FIG. 6A),WZ141-90 (FIG. 6B), and WZ141-91 (FIG. 6C) inhibit Nluc in a dose- andtime-dependent manner.

FIGS. 7A-7F show the permeability of the thienopyrrole compoundsJRW-0138 (FIG. 7A), JRW-0140 (FIG. 7B), JRW-0142 (FIG. 7C), JRW-0143(FIG. 7D), JRW-0145 (FIG. 7E), and JRW-0147 (FIG. 7F) using HEK293 orHeLa cells transiently transfected with a Beta-2 AdrenergicReceptor-Nluc fusion protein.

FIGS. 8A-8F show the permeability of the thienopyrrole compoundsJRW-0148 (FIG. 8A), JRW-0149 (FIG. 8B), JRW-0151 (FIG. 8C), JRW-0152(FIG. 8D), JRW-0051 (FIG. 8E), and JRW-0043 (FIG. 8F) using HEK293 orHeLa cells transiently transfected with a Beta-2 AdrenergicReceptor-Nluc fusion protein.

FIGS. 9A-9F show the permeability of the thienopyrrole compoundsJRW-0044 (FIG. 9A), JRW-0013 (FIG. 9B), JRW-0034 (FIG. 9C), JRW-0052(FIG. 9D), JRW-0110 (FIG. 9E), and JRW-0187 (FIG. 9F) using HEK293 orHeLa cells transiently transfected with a Beta-2 AdrenergicReceptor-Nluc fusion protein.

FIGS. 10A-10F show the permeability of the thienopyrrole compoundsJRW-0188 (FIG. 10A), JRW-0190 (FIG. 10B), JRW-0191 (FIG. 10C), JRW-0192(FIG. 10D), JRW-0195 (FIG. 10E), and JRW-0197 (FIG. 10F) using HEK293 orHeLa cells transiently transfected with a Beta-2 AdrenergicReceptor-Nluc fusion protein.

FIGS. 11A-11C show the permeability of the thienopyrrole compoundsJRW-0198 (FIG. 11A), JRW-0200 (FIG. 11B), and JRW-0208 (FIG. 11C) usingHEK293 or HeLa cells transiently transfected with a Beta-2 AdrenergicReceptor-Nluc fusion protein.

FIGS. 12A-12E show the ability of the thienopyrrole compounds to inhibitextracellular BRET. FIG. 12A shows a schematic diagram of spuriousextracellular BRET assay design. FIGS. 12B-12E show compound responsecurves of the thienopyrrole compounds, JRW-0013 (FIG. 12B), JRW-0051(FIG. 12C), JRW-0147 (FIG. 12D), and JRW-0187 (FIG. 12E) using theextracellular BRET assay.

FIGS. 13A-13E show the ability of the thienopyrrole compounds to inhibitextracellular luciferase activity and enhance intracellular BRET. FIG.13A shows a schematic diagram of spurious extracellular Nluc assaydesign. FIGS. 13B-13E show compound response curves of the thienopyrrolecompounds, JRW-0013 (FIG. 13B), JRW-0051 (FIG. 13C), JRW-0147 (FIG.13D), and JRW-0187 (FIG. 13E) using the extracellular Nluc assay.

FIG. 14 shows the cell permeability of thienopyrrole compounds JRW-0147,JRW-0051, and JRW-0138.

FIGS. 15A-15C show the cell impermeability of thienopyrrole compoundJRW-0147 in a target engagement model. FIG. 15A shows a schematicdiagram of SRC-Nluc assay design. FIGS. 15B-13C show compound responsecurves for Dasatinib-DY607 (FIG. 15B) and JRW-0147 (FIG. 15C).

FIG. 16 shows the permeability of the thienopyrrole compounds JRW-0147and JRW-0013.

FIGS. 17A-17C show the permeability of the thienopyrrole compoundsJRW-0013 (FIG. 17A), JRW-0147 (FIG. 17B), and JRW-0344 (FIG. 17C) usingHEK293 cells transiently transfected with a Beta-2 AdrenergicReceptor-Nluc fusion protein.

FIGS. 18A-18C show the cell viability and toxicity of JRW-0344 (FIG.18A) compared to digitonin (FIG. 18B) and DMSO (FIG. 18C).

FIG. 19 shows the cell permeability of thienopyrrole compounds JRW-0147,JRW-0344, and JRW-0013.

FIGS. 20A-20C show the ability of the thienopyrrole compound JRW-0344 toinhibit extracellular luciferase activity. FIG. 20A shows a schematicdiagram of the SRC-Nluc assay design. FIGS. 20B and 20C show compoundresponse curves of the thienopyrrole compound JRW-0344 using theSRC-Nluc assay (FIG. 20B) or extracellular Nluc assay (FIG. 20C).

DETAILED DESCRIPTION

Disclosed herein are thienopyrrole compounds that can selectivelyinhibit Oplophorus-derived luciferases, such as a luciferase of SEQ IDNO:2 (also interchangeably referred to herein as “NanoLuc”, “Nluc,”“Nluc luciferase,” and “Nluc enzyme”). Due to their stabilities andtheir potential to be excreted from cells, it may be advantageous to useselective inhibitors to suppress the luminescence fromOplophorus-derived luciferases in certain applications. For example, inapplications involving temporal multiplexing of multiple luminescentsystems, it can be beneficial to have selective inhibitors for eachsystem to allow for the measurement and/or detection of only oneluminescent signal at a time. Additionally, in some plate-based assays,a certain amount of luciferase may be excreted from cells. Anextracellular inhibitor compound would allow for luminescence fromexcreted luciferase to be selectively suppressed and may, therefore,help to improve the signal-to-noise ratio in certain assays.

Thienopyrrole compounds described herein have been found to be selectiveinhibitors for Oplophorus luciferases. The thienopyrrole compounds maycompete for binding of the coelenterazine substrates of the luciferasesand can be modified to produce both cell-permeable and cell-impermeableinhibitors.

1. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

As used herein, the term “substituent” or “suitable substituent” isintended to mean a chemically acceptable functional group e.g., a moietythat does not negate the activity of the inventive compounds.Illustrative examples of suitable substituents include, but are notlimited to halo groups, perfluoroalkyl groups, perfluoroalkoxy groups,alkyl groups, alkenyl groups, alkynyl groups, hydroxy groups, halogroups, oxo groups, mercapto groups, alkylthio groups, alkoxy groups,nitro groups, azidealkyl groups, sulfonic acid groups, aryl orheteroaryl groups, aryloxy or heteroaryloxy groups, aralkyl orheteroaralkyl groups, aralkoxy or heteroaralkoxy groups, HO—(C═O)—groups, heterocylic groups, cycloalkyl groups, amino groups, alkyl- anddialkyl-amino groups, carbamoyl groups, alkylcarbonyl groups,alkylcarbonyloxy groups, alkoxycarbonyl groups, alkylaminocarbonylgroups, dialkylamino carbonyl groups, arylcarbonyl groups,aryloxycarbonyl groups, alkylsulfonyl groups, arylsulfonyl groups andthe like. The substituents can be substituted by additionalsubstituents. The substituents can also be in salt forms (e.g., asulfonic acid group can be in the form of a sulfonate group.

As used herein, the term “alkenyl” refers a straight or branchedhydrocarbon chain containing from 2 to 10 carbons and containing atleast one carbon-carbon double bond formed by the removal of twohydrogens. Representative examples of alkenyl include, but are notlimited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl,4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.Alkenyl groups of the present invention may be unsubstituted orsubstituted by one or more suitable substituents, preferably 1 to 3suitable substituents, as defined above.

As used herein, the term “alkoxy” refers to an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy.

As used herein, the term “alkoxyalkoxy” refers to an alkoxy group, asdefined herein, appended to the parent molecular moiety through anotheralkoxy group, as defined herein. Representative examples of alkoxyalkoxyinclude, but are not limited to, tert-butoxymethoxy, 2-ethoxyethoxy,2-methoxyethoxy, and methoxymethoxy.

The term “alkoxyalkoxyalkyl” as used herein, means an alkoxyalkoxygroup, as defined herein, appended to the parent molecular moietythrough an alkylene group, as defined herein. Representative examples ofalkoxyalkoxyalkyl include, but are not limited to,tert-butoxymethoxymethyl, ethoxymethoxymethyl, (2-methoxyethoxy)methyl,and 2-(2-methoxyethoxy)ethyl.

As used herein, the term “alkoxyalkyl” refers to an alkoxy group, asdefined herein, appended to the parent molecular moiety through an alkylgroup, as defined herein. Representative examples of alkoxyalkylinclude, but are not limited to, tert-butoxymethyl, 2-ethoxyethyl,2-methoxyethyl, and methoxymethyl.

As used herein, the term “alkoxycarbonyl” refers to an alkoxy group, asdefined herein, appended to the parent molecular moiety through acarbonyl group, as defined herein. Representative examples ofalkoxycarbonyl include, but are not limited to, methoxycarbonyl,ethoxycarbonyl, and tert-butoxycarbonyl.

The term “alkoxycarbonylalkyl” as used herein, means an alkoxycarbonylgroup, as defined herein, appended to the parent molecular moietythrough an alkylene group, as defined herein. Representative examples ofalkoxycarbonylalkyl include, but are not limited to,ethoxycarbonylmethyl, 3-methoxycarbonylpropyl, 4-ethoxycarbonylbutyl,and 2-tert-butoxycarbonylethyl.

As used herein, the term “alkyl” refers to a linear or branchedhydrocarbon radical, suitably having 1 to 30 carbon atoms, 1 to 12carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbonatoms, or 1 to 4 carbon atoms. The term “C₁-C₈-alkyl” is defined toinclude alkyl groups having 1, 2, 3, 4, 5, 6, 7 or 8 carbons in a linearor branched arrangement. For example, “C₁-C₈-alkyl” specificallyincludes methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl,iso-butyl, tert-butyl, pentyl (e.g., n-pentyl), hexyl (e.g., n-hexyl),heptyl (e.g., n-heptyl) and octyl (e.g., n-octyl). The term“C₁-C₆-alkyl” is defined to include alkyl groups having 1, 2, 3, 4, 5,or 6 carbons in a linear or branched arrangement. For example,“C₁-C₆-alkyl” specifically includes methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tert-butyl, pentyl (e.g., n-pentyl), andhexyl (e.g., n-hexyl). The term “C₁-C₄-alkyl” is defined to includealkyl groups having 1, 2, 3, or 4 carbons in a linear or branchedarrangement. For example, “C₁-C₄-alkyl” specifically includes methyl,ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, iso-butyl and tert-butyl.Alkyl groups of the present invention may be unsubstituted orsubstituted by one or more suitable substituents, such as 1 to 3suitable substituents, as defined above. For example, an alkyl group canbe substituted with one or more halo substituents to form a haloalkylgroup, or with one or more hydroxy substituents to form a hydroxyalkylgroup, or with one or more alkoxy groups to form an alkoxyalkyl group.

As used herein, the term “alkylamino” refers to an alkyl group, asdefined herein, appended to the parent molecular moiety through an aminogroup, as defined herein. Representative examples of alkylamino include,but are not limited to, methylamino, ethylamino, iso-propylamino,butyl-amino and sec-butylamino.

As used herein, the term “alkylaminoalkyl” refers to an alkyl group, asdefined herein, appended to the parent molecular moiety through anaminoalkyl group, as defined herein. Representative examples ofalkylaminoalkyl groups include, but are not limited to, methylaminoethyland methylamino-2-propyl.

As used herein, the term “alkylcarbonyl” refers to an alkyl group, asdefined herein, appended to the parent molecular moiety through acarbonyl group, as defined herein. Representative examples ofalkylcarbonyl include, but are not limited to, acetyl, 1-oxopropyl,2,2-dimethyl-1-oxopropyl, 1-oxobutyl, and 1-oxopentyl.

As used herein, the term “alkylcarbonylalkyl” refers to an alkylcarbonylgroup, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein.

As used herein, the term “alkylcarbonylalkylamido” refers to analkylcarbonyl group, as defined herein, appended to the parent molecularmoiety through an alkylamido group, as defined herein.

The term “alkylene” means a divalent group derived from a saturated,straight or branched chain hydrocarbon of from 1 to 10 carbon atoms.Representative examples of alkylene include, but are not limited to,—CH₂—, —CH(CH₃)—, —C(CH₃)₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—,—CH₂C(CH₃)₂—, and —CH₂CH(CH₃)CH₂—.

As used herein, the term “alkynyl” refers to a straight or branchedhydrocarbon radical having 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons, andhaving one or more carbon-carbon triple bonds. Alkynyl groups of thepresent invention include, but are not limited to, ethynyl, propynyl,and butynyl. Alkynyl groups of the present invention may beunsubstituted or substituted by one or more suitable substituents,preferably 1 to 3 suitable substituents, as defined above.

As used herein, the term “amido” refers to an amino group appended tothe parent molecular moiety through a carbonyl group, as defined herein(i.e., —CONH₂). The term “alkylamido,” as used herein, refers to analkylamino group or dialkylamino group appended to the parent molecularmoiety through a carbonyl group, as defined herein. Representativeexamples of alkylamido include, but are not limited to,methylaminocarbonyl, dimethylaminocarbonyl, ethylmethylaminocarbonyl,and n-hexylaminocarbonyl.

As used herein, the term “amino” refers to an —NH₂ group.

As used herein, the term “aminoalkyl” refers to at least one aminogroup, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein. Representative examples ofaminoalkyl include, but are not limited to, aminomethyl, 2-aminoethyl,3-aminopropyl, 4-aminobutyl, 5-aminopentyl, and 6-aminohexyl.

As used herein, the term “aminoalkylamido” refers to at least one aminogroup, as defined herein, appended to the parent molecular moietythrough an alkylamido group, as defined herein.

As used herein, the term “amino protecting group,” refers to a moietythat prevents chemical reactions from occurring on the nitrogen atom towhich that protecting group is attached. An amino protecting group mustalso be removable by a chemical reaction. Such groups are well known inthe art and include those described in detail in Protecting Groups inOrganic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, JohnWiley & Sons, 1999, the entirety of which is incorporated herein byreference. Suitable amino protecting groups include, but are not limitedto, carbobenzyloxy (—NHCO—OCH₂C₆H₅ or —NH-Cbz); t-butyloxycarbonyl(—NHCO—OC(CH₃)₃ or —NH-Boc); 9-fluorenylmethyloxycarbonyl (—NH-Fmoc),2,2,2-trichloroethyloxycarbonyl (—NH-Troc), and allyloxycarbonyl(—NH-Alloc). (In each of the above, the —NH— represents the nitrogenfrom the amino group that is being protected.)

As used herein, the term “aminoluciferin” refers to(4S)-2-(6-amino-1,3-benzothiazol-2-yl)-4,5-dihydrothiazole-4-carboxylicacid, or a substituted analog of this molecule.

As used herein, the term “aryl” means monocyclic, bicyclic, or tricyclicaromatic radicals. Representative examples of the aryl groups include,but are not limited to, phenyl, dihydroindenyl, indenyl, naphthyl,dihydronaphthalenyl, and tetrahydronaphthalenyl. Aryl groups of thepresent invention may be optionally substituted by one or more suitablesubstituents, preferably 1 to 5 suitable substituents, as defined above.

As used herein, the term “arylalkyl” refers to an aryl group, as definedherein, appended to the parent molecular moiety through an alkyl group,as defined herein. Representative examples of arylalkyl include, but arenot limited to, phenylmethyl (i.e. benzyl) and phenylethyl.

As used herein, the term “arylcarbonyl” refers to an aryl group, asdefined herein, appended to the parent molecular moiety through acarbonyl group, as defined herein.

As used herein, the term “bioluminescence” or “luminescence” may referto light produced as a result of a reaction between an enzyme and asubstrate that generates light. Examples of such enzymes (bioluminescentenzymes) include Oplophorus luciferase, e.g., Oplophorousgracilirostris, firefly luciferase, e.g. Photinus pyralis or Photurispennsylvanica, click beetle luciferase, Renilla luciferase, cypridinaluciferase, Aequorin photoprotein, obelin photoprotein and the like.

As used herein, the term “carbonyl” or “(C═O)” (as used in phrases suchas alkylcarbonyl, alkyl-(C═O)— or alkoxycarbonyl) refers to the joinderof the >C═O moiety to a second moiety such as an alkyl or amino group(i.e. an amido group). Alkoxycarbonylamino (i.e. alkoxy(C═O)—NH—) refersto an alkyl carbamate group. The carbonyl group is also equivalentlydefined herein as (C═O). Alkylcarbonylamino refers to groups such asacetamide.

As used herein, the term “carboxy” refers to a —C(O)OH group.

As used herein, the term “carboxyalkyl” refers to a carboxy group asdefined herein, appended to the parent molecular moiety through an alkylgroup as defined herein.

As used herein, the term “carboxyalkylamido” refers to a carboxyalkylgroup as defined herein, appended to the parent molecular moiety throughan amido group as defined herein.

As used herein, the term “coelenterazine substrate” refers to a class ofreporter molecules that luminesce when acted upon by a wide variety ofbioluminescent proteins such as luciferases (e.g., marine luciferases).Coelenterazine substrates include coelenterazine as well as analogs andderivatives thereof.

As used herein, the term “cycloalkyl” refers to a mono, bicyclic ortricyclic carbocyclic radical (e.g., cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl,cyclopentenyl, cyclohexenyl, bicyclo[2.2.1]heptanyl,bicyclo[3.2.1]octanyl and bicyclo[5.2.0]nonanyl, etc.); optionallycontaining 1 or 2 double bonds. Cycloalkyl groups of the presentinvention may be unsubstituted or substituted by one or more suitablesubstituents, preferably 1 to 5 suitable substituents, as defined above.

As used herein, the term “cycloalkylalkyl” refers to a cycloalkyl group,as defined herein, appended to the parent molecular moiety through analkyl group, as defined herein. Representative examples ofcycloalkylalkyl include, but are not limited to, cyclohexylmethyl.

As used herein, the term “cycloalkylamido” refers to a cycloalkyl group,as defined herein, appended to the parent molecular moiety through anamido group, as defined herein.

As used herein, the term “dialkylamino” refers to two independentlyselected alkyl groups, as defined herein, appended to the parentmolecular moiety through an amino group, as defined herein.Representative examples of dialkylamino include, but are not limited to,N,N-dimethylamino, N-ethyl-N-methylamino, and N-isopropyl-N-methylamino.

As used herein, the term “dialkylaminoalkyl” refers to a dialkylaminogroup, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein. Representative examples ofdialkylaminoalkyl include, but are not limited to,N,N-dimethylaminoethyl and N,N-methyl(2-propyl)aminoethyl.

As used herein, the term “dialkylaminoalkylamido” refers to adialkylamino group, as defined herein, appended to the parent molecularmoiety through an alkylamido group, as defined herein.

As used herein, the term “halogen” or “halo” refers to a fluoro, chloro,bromo or iodo radical.

As used herein, the term “haloalkoxy” refers to an alkoxy group, asdefined herein, substituted by one, two, three, or four halogen atoms.Representative examples of haloalkoxy include, but are not limited to,chloromethoxy, 2-fluoroethoxy, trifluoromethoxy, and pentafluoroethoxy.

As used herein, the term “haloalkyl” refers to an alkyl group, asdefined herein, substituted by one, two, three, or four halogen atoms.Representative examples of haloalkyl include, but are not limited to,chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl,2-chloro-3-fluoropentyl, and 4,4,4,-trifluorobutyl.

As used herein, the term “heteroaryl” refers to a monocyclic heteroarylor a bicyclic heteroaryl. The monocyclic heteroaryl is a five- orsix-membered ring. The five-membered ring contains two double bonds. Thefive-membered ring may contain one heteroatom selected from O or S; orone, two, three, or four nitrogen atoms and optionally one oxygen orsulfur atom. The six-membered ring contains three double bonds and one,two, three or four nitrogen atoms. Representative examples of monocyclicheteroaryl include, but are not limited to, furanyl, imidazolyl,isoxazolyl, isothiazolyl, oxadiazolyl, 1,3-oxazolyl, pyridinyl,pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl,thiadiazolyl, 1,3-thiazolyl, thienyl, triazolyl, and triazinyl. Thebicyclic heteroaryl includes a monocyclic heteroaryl fused to a phenyl,or a monocyclic heteroaryl fused to a monocyclic cycloalkyl, or amonocyclic heteroaryl fused to a monocyclic cycloalkenyl, or amonocyclic heteroaryl fused to a monocyclic heteroaryl, or a monocyclicheteroaryl fused to a monocyclic heterocycle. Representative examples ofbicyclic heteroaryl groups include, but are not limited to,benzofuranyl, benzothienyl, benzoxazolyl, benzimidazolyl,benzoxadiazolyl, 6,7-dihydro-1,3-benzothiazolyl,imidazo[1,2-a]pyridinyl, indazolyl, indolyl, isoindolyl, isoquinolinyl,naphthyridinyl, pyridoimidazolyl, quinazolinyl, quinolinyl,thiazolo[5,4-b]pyridin-2-yl, thiazolo[5,4-d]pyrimidin-2-yl, and5,6,7,8-tetrahydroquinolin-5-yl. Heteroaryl groups of the presentinvention may be unsubstituted or substituted by one or more suitablesubstituents, preferably 1 to 5 suitable substituents, as defined above.

As used herein, the term “heteroarylalkyl” refers to a heteroaryl group,as defined herein, appended to the parent molecular moiety through analkyl group, as defined herein. Representative examples ofheteroarylalkyl include, but are not limited to, fur-3-ylmethyl,1H-imidazol-2-ylmethyl, 1H-imidazol-4-ylmethyl, 1-(pyridin-4-yl)ethyl,pyridin-3-ylmethyl, 6-chloropyridin-3-ylmethyl, pyridin-4-ylmethyl,(6-(trifluoromethyl)pyridin-3-yl)methyl, (6-(cyano)pyridin-3-yl)methyl,(2-(cyano)pyridin-4-yl)methyl, (5-(cyano)pyridin-2-yl)methyl,(2-(chloro)pyridin-4-yl)methyl, pyrimidin-5-ylmethyl,2-(pyrimidin-2-yl)propyl, thien-2-ylmethyl, and thien-3-ylmethyl.

As used herein, the term “heterocycle” or “heterocyclyl” refers to amonocyclic heterocycle, a bicyclic heterocycle, or a tricyclicheterocycle. The monocyclic heterocycle is a three-, four-, five-, six-,seven-, or eight-membered ring containing at least one heteroatomindependently selected from the group consisting of oxygen, nitrogen,phosphorus and sulfur. The three- or four-membered ring contains zero orone double bond, and one heteroatom selected from the group consistingof oxygen, nitrogen, phosphorus and sulfur. The five-membered ringcontains zero or one double bond and one, two or three heteroatomsselected from the group consisting of oxygen, nitrogen, phosphorus andsulfur. The six-membered ring contains zero, one or two double bonds andone, two, or three heteroatoms selected from the group consisting ofoxygen, nitrogen, phosphorus and sulfur. The seven- and eight-memberedrings contains zero, one, two, or three double bonds and one, two, orthree heteroatoms selected from the group consisting of oxygen,nitrogen, phosphorus and sulfur. Representative examples of monocyclicheterocycles include, but are not limited to, azetidinyl, azepanyl,aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl,1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl,isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl,oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, phosphinane,piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl,pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothienyl,thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl,thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone),thiopyranyl, trithianyl, and 2,5-dioxo-pyrrolidinyl. The bicyclicheterocycle is a monocyclic heterocycle fused to a phenyl group, or amonocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclicheterocycle fused to a monocyclic cycloalkenyl, or a monocyclicheterocycle fused to a monocyclic heterocycle, or a bridged monocyclicheterocycle ring system in which two non-adjacent atoms of the ring arelinked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or analkenylene bridge of two, three, or four carbon atoms. Representativeexamples of bicyclic heterocycles include, but are not limited to,benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl,2,3-dihydrobenzothienyl, azabicyclo[2.2.1]heptyl (including2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl,octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl,9-phosphabicyclo[3.3.1]nonane, 8-phosphabicyclo[3.2.1]octane, andtetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by abicyclic heterocycle fused to a phenyl group, or a bicyclic heterocyclefused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to amonocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclicheterocycle, or a bicyclic heterocycle in which two non-adjacent atomsof the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4carbon atoms, or an alkenylene bridge of two, three, or four carbonatoms. Examples of tricyclic heterocycles include, but are not limitedto, octahydro-2,5-epoxypentalene,hexahydro-2H-2,5-methanocyclopenta[b]furan,hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-admantane(1-azatricyclo[3.3.1.1^(3,7)]decane), oxa-adamantane(2-oxatricyclo[3.3.1.1^(3,7)]decane), and2,4,6-trioxa-8-phosphatricyclo[3.3.1.13,7]decane. Heterocyclic groups ofthe present invention may be unsubstituted or substituted by one or moresuitable substituents, preferably 1 to 3 suitable substituents, asdefined above. Heterocyclic groups of the present invention may be cancontain one or more oxo groups (═O) or thioxo (═S) groups attached tothe ring.

As used herein, the term “heterocyclylalkyl” refers to a heterocyclylgroup, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein. Representative examples ofheterocyclylalkyl include, but are not limited to, piperidin-4-ylmethyl,piperazin-1-ylmethyl, 3-methyl-1-pyrrolidin-1-ylbutyl,(1R)-3-methyl-1-pyrrolidin-1-ylbutyl,(1S)-3-methyl-1-pyrrolidin-1-ylbutyl, and 3-morpholinopropyl.

As used herein, the term “heterocyclylamido” refers to a heterocyclylgroup, as defined herein, appended to the parent molecular moietythrough an amido group, as defined herein.

As used herein, the term “hydroxy” refers to an —OH group.

As used herein, the term “hydroxyalkoxy” refers to an alkoxy group, asdefined herein, substituted by at least one hydroxy group.Representative examples of hydroxyalkoxy include, but are not limitedto, hydroxyethoxy, and 2-hydroxypropoxy.

As used herein, the term “hydroxyalkyl” refers to an alkyl group, asdefined herein, substituted by at least one hydroxy group.Representative examples of hydroxyalkyl include, but are not limited to,hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl,2,3-dihydroxypentyl, 4-hydroxybutyl, 2-ethyl-4-hydroxyheptyl,3,4-dihydroxybutyl, and 5-hydroxypentyl.

The term “hydroxyalkylamido” as used herein refers to a hydroxyalkylgroup attached to an amido group, e.g., -amido-alkyl-OH.

As used herein, the term “hydroxycarbonyl” refers to a hydroxy group, asdefined herein, appended to the parent molecular moiety through acarbonyl group, as defined herein.

The term “luminescent enzyme,” “bioluminescent enzyme,” or “luciferase”as used interchangeably herein refers to a class of oxidative enzymesused in bioluminescence wherein the enzyme produces and emits light whengiven a substrate. The luciferase may be a naturally occurring,recombinant, or mutant luciferase that uses a luciferase substrate. Theluciferase substrate may be luciferin, a luciferin derivative or analog,a preluciferin derivative or analog, a coelenterazine, or acoelenterazine derivative or analog. The luminescent enzyme, ifnaturally occurring, may be obtained easily by the skilled person froman organism. If the luminescent enzyme is one that occurs naturally oris a recombinant or mutant luminescent enzyme, e.g. one which retainsactivity in a luciferase-coelenterazine or luciferase-luciferin reactionof a naturally occurring luminescent enzyme, it can be obtained readilyfrom a culture of bacteria, yeast, mammalian cells, insect cells, plantcells, or the like, transformed to express a nucleic acid encoding theluminescent enzyme. Further, the recombinant or mutant luminescentenzyme can be derived from an in vitro cell-free system using a nucleicacid encoding the luciferase. Suitable luminescent enzymes includeluciferases derived from bioluminescent decapods, such as from theOplophoroidea (e.g. Oplophorus-derived luciferases), beetle luciferases(e.g., Photinus pyralis, Photuris pennsylvanica, etc.), marine organismssuch as cnidarians (e.g., Renilla luciferase), Aristeidae,Solenoceridae, Luciferidae, Sergestidae, Pasipheidae andThalassocarididae decapoda families, copepod luciferases, such asGaussia luciferase, such as Gaussia princeps luciferase, Metridialuciferases, such as Metridia longa and Metridia pacifica luciferases,Vargula luciferases, such as Vargula hilgendorfii luciferase,Pleuromamma xiphias luciferase, and photoproteins, such as Aequorin, andvariants, recombinants, and mutants thereof.

A “luminescent reaction mixture” contains materials that will allow theluminescent enzyme to generate a light signal, i.e., luminescence. Themixture may also contain the enzyme, e.g., the luciferase enzyme orluciferase. The materials, and the particular concentrations and/oramounts, needed to generate a luminescent signal will vary depending onthe luminescent enzyme used as well as the type of assay beingperformed. Often other materials will be added to the solutionincluding: a buffer to maintain the reaction at the proper pH, anadditive such as PRIONEX or Bovine serum albumin (BSA) to help maintainenzyme activity, reducing agents, detergents, etc.

As used herein, the term “methylenedioxy” refers to a —OCH₂O— groupwherein the oxygen atoms of the methylenedioxy are attached to theparent molecular moiety through two adjacent carbon atoms.

As used herein, the terms “Oplophorus luciferase” and“Oplophorus-derived luciferase” are used interchangeably and refer to aluciferase secreted from the deep-sea shrimp Oplophorus gracihrostris(e.g., SEQ ID NO: 1), including wild-type, variants, and mutantsthereof. For example, suitable Oplophorus luciferase variants aredescribed in U.S. Pat. Nos. 8,557,970 and 8,669,103, each of which isincorporated herein by reference in its entirety. ExemplaryOplophorus-derived luciferases include, for example, that of SEQ ID NO:2 (also interchangeably referred to herein as “NanoLuc”, “Nluc,” “Nlucluciferase,” and “Nluc enzyme”).

As used herein, the term “oxo” refers to a double bonded oxygen (═O)radical wherein the bond partner is a carbon atom. Such a radical canalso be thought as a carbonyl group.

The term “peptide” or “polypeptide” refers to a sequence of at least twoamino acids. In some embodiments, a peptide may contain no more than 80amino acids, or no more than 35 amino acids, no more than 10 aminoacids, or no more than 5 amino acids.

As used herein, the term “reporter moiety” may refer to a moiety that,under appropriate conditions, directly or indirectly generates adetectable signal. Exemplary reporter moieties include, but are notlimited to, fluorophores, luminescent molecules, dyes, radiolabels andsubstrates for enzymes such as luciferase. In some embodiments, areporter moiety may indirectly generate a detectable signal, forexample, when the reporter moiety is a substrate for an enzyme. Thereaction of the enzyme with the substrate then produces a detectablesignal such as fluorescence or luminescence. As used herein, the term“bioluminescent reporter moiety” may refer to a moiety that is asubstrate for a luciferase. For example, the bioluminescent reportermoiety can be a luciferin, a luciferin derivative, e.g., pre-luciferin,aminoluciferin, quionolyl-luciferin, napthyl luciferin, fluorolucifeirn,chloroluciferin, precursors of luciferin derivatives, a coelenterazineor a coelenterazine derivative or analog, e.g., furimazine. Theluminescent signal generated may be detected using a luminometer. Asused herein, the term “fluorescent reporter moiety” may refer to amoiety that fluoresces. For example, the fluorescent reporter moiety maybe a flurophore, such as coumarin, R110, fluoroscein, DDAO, resorufin,cresyl violet, sily xanthene, or carbopyronine. Fluorescence may bedetected using a fluorometer.

A prefix attached to a multi-component substituent only applies to thefirst component it precedes. To illustrate, the term “cycloalkylalkyl”contains two components: alkyl and cycloalkyl. Thus, the C₁-C₆-prefix onC₁-C₆-cycloalkylalkyl means that the alkyl component of thecycloalkylalkyl contains from 1 to 6 carbon atoms; the C₁-C₆-prefix doesnot describe the cycloalkyl component. To illustrate further, the term“halo-C₁-C₆-alkyl” refers to halomethyl, haloethyl, halopropyl,halobutyl, halopentyl, and halohexyl. To illustrate further, the prefix“halo” on haloalkoxyalkyl indicates that only the alkoxy component ofthe alkoxyalkyl substituent is substituted with one or more halogenradicals. If the halogen substitution may only occur on the alkylcomponent, the substituent would instead be described as“alkoxyhaloalkyl.”

A substituent is “substitutable” if it comprises at least one carbon ornitrogen atom that is bonded to one or more hydrogen atoms. Thus, forexample, hydrogen, halogen, and cyano do not fall within thisdefinition. In addition, a sulfur atom in a heterocyclyl containing suchatom is substitutable with one or two oxo substituents.

If a substituent is described as being “substituted,” a non-hydrogenradical is in the place of hydrogen radical on a carbon or nitrogen ofthe substituent. Thus, for example, a substituted alkyl substituent isan alkyl substituent in which at least one non-hydrogen radical is inthe place of a hydrogen radical on the alkyl substituent. To illustrate,monofluoroalkyl is alkyl substituted with a fluoro radical, anddifluoroalkyl is alkyl substituted with two fluoro radicals. It shouldbe recognized that if there is more than one substitution on asubstituent, each non-hydrogen radical may be identical or different(unless otherwise stated).

When a substituent is referred to as “unsubstituted” or not referred toas “substituted” or “optionally substituted,” it means that thesubstituent does not have any substituents. If a substituent isdescribed as being “optionally substituted,” the substituent may beeither (1) unsubstituted or (2) substituted. If a substituent isdescribed as being optionally substituted with up to a particular numberof non-hydrogen radicals, that substituent may be either (1)unsubstituted; or (2) substituted by up to that particular number ofnon-hydrogen radicals or by up to the maximum number of substitutablepositions on the substituent, whichever is less. Thus, for example, if asubstituent is described as a heteroaryl optionally substituted with upto 3 non-hydrogen radicals, then any heteroaryl with less than 3substitutable positions would be optionally substituted by up to only asmany non-hydrogen radicals as the heteroaryl has substitutablepositions. To illustrate, tetrazolyl (which has only one substitutableposition) would be optionally substituted with up to one non-hydrogenradical. To illustrate further, if an amino nitrogen is described asbeing optionally substituted with up to 2 non-hydrogen radicals, then aprimary amino nitrogen will be optionally substituted with up to 2non-hydrogen radicals, whereas a secondary amino nitrogen will beoptionally substituted with up to only 1 non-hydrogen radical.

If substituents are described as being “independently selected” from agroup, each substituent is selected independent of the other. Eachsubstituent, therefore, may be identical to or different from the othersubstituent(s).

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

2. THIENOPYRROLE COMPOUNDS

Provided herein are thienopyrrole compounds that may inhibitOplophorus-derived luciferases and/or Oplophorus-derived luciferaseactivity. The thienopyrrole compounds include compounds of formula (I)and salts thereof:

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl; and

R³ and R⁴ are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring.

In some embodiments, the dashed line represents the presence of a bond,and X is CH.

In some embodiments, n is 1.

In some embodiments, A is a phenyl ring. In some embodiments, A is a5-membered heteroaryl ring. In some embodiments, A is a thienyl ring. Insome embodiments, A is a furanyl ring.

In some embodiments, R¹ is selected from the group consisting ofhydrogen, C₁-C₈ alkyl, halo-C₁-C₈-alkyl, alkoxyalkoxyalkyl andarylalkyl. For example, in some embodiments, R¹ is selected from thegroup consisting of hydrogen, ethyl, n-hexyl, 2-(2-methoxyethoxy)ethyland benzyl. In some embodiments, R¹ is ethyl.

In some embodiments, R² is optionally substituted aryl. For example, insome embodiments, R² is substituted phenyl. R² is phenyl substitutedwith one substituent selected from the group consisting of C₁-C₄ alkyl,cyano, amido, C₁-C₄ alkoxy, and hydroxyalkyl. In some embodiments, R² isphenyl substituted with one methyl group (e.g., m-tolyl).

In some embodiments, R¹ is C₁-C₄ alkyl and R² is optionally substitutedphenyl. In some embodiments, R¹ is ethyl and R² is m-tolyl.

In some embodiments, R³ and R⁴, together with the nitrogen atom to whichthey are attached, together form an optionally substituted ring. In someembodiments, R³ and R⁴, together with the nitrogen atom to which theyare attached, together form an optionally substituted monocyclicheterocycle. In some embodiments, the optionally substituted monocyclicheterocycle is selected from the group consisting of optionallysubstituted pyrrolidine, piperidine and piperazine. In some embodiments,the optionally substituted monocyclic heterocycle is selected from thegroup consisting of unsubstituted pyrrolidine, unsubstituted piperidine,piperidine substituted with one substituent (e.g., alkoxycarbonyl suchas ethoxycarbonyl), or piperazine substituted with one substituent(e.g., C₁-C₄ alkyl such as methyl).

In some embodiments, R³ is hydrogen.

In some embodiments, R⁴ is selected from the group consisting ofunsubstituted C₁-C₈ alkyl (e.g., methyl), halo-C₁-C₈-alkyl,carboxy-C₁-C₈-alkyl (e.g., —(CH₂)₇—COOH),C₁-C₄-alkoxycarbonyl-C₁-C₈-alkyl (e.g., —(CH₂)₇—COOCH₃), optionallysubstituted phenyl (below), optionally substituted C₅-C₆ cycloalkyl(e.g., unsubstituted cyclopentyl, unsubstituted cyclohexyl, orsubstituted cyclohexyl), optionally substituted heterocyclyl (e.g.,unsubstituted piperidinyl, piperidinyl substituted withtert-butoxycarbonyl, alkoxycarbonylalkylcarbonyl such as—CO—(CH₂)₄—COOCH₃), or carboxyalkylcarbonyl such as —CO—(CH₂)₄—COOH),optionally substituted heteroarylalkyl (e.g., pyridyl-C₁-C₄-alkyl suchas —CH₂-pyridyl), and optionally substituted heterocyclylalkyl (e.g.,morpholino-C₁-C₄-alkyl such as —(CH₂)₃-morpholino). For example, in someembodiments, R⁴ is phenyl that is unsubstituted or substituted with onesubstituent, such as a substituent selected from the group consisting ofC₁-C₄-alkoxycarbonyl (e.g., —C(O)OCH₃) andC₁-C₄-alkoxycarbonyl-C₁-C₄-alkyl (e.g., —CH₂—C(O)CH₂CH₃).

In some embodiments, R⁴ is cyclohexyl substituted with one substituentselected from the group consisting of carboxy, C₁-C₄-alkoxycarbonyl(e.g., —C(O)OCH₃), C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₃CH₃ or—C(O)NH—(CH₂)₅CH₃), hydroxy-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₂OH or—C(O)NH—(CH₂)₆OH), amido (i.e. —CONH₂), optionally substitutedamino-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₆NH₂),C₁-C₄-dialkylamino-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₂N(CH₃)₂),carboxy-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₃COOH, —C(O)NH—(CH₂)₅COOHor —C(O)NH—(CH₂)₇COOH), sulfonic acid-C₁-C₈-alkylamido (e.g.,—C(O)NH—(CH₂)₆SO₃H), sulfonate-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₆SO₃⁻), C₁-C₄-alkylcarbonyl-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₃COOCH₃,—C(O)NH—(CH₂)₅COOCH₃ or —C(O)NH—(CH₂)₇COOCH₃), optionally substitutedC₃-C₆-cycloalkylamido (e.g., —C(O)NH-cyclohexyl, —C(O)NH-cyclohexyl-COOHor —C(O)NH-cyclohexyl-COOCH₃), and heterocyclylamido (e.g.,—C(O)NH-piperidinyl, unsubstituted or substituted with methyl,tert-butoxycarbonyl or acetyl). For example, in some embodiments, R⁴ isoptionally substituted amino-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₆NH₂),wherein the amino group is protected with an amino protecting group(e.g., tert-butoxycarbonyl), or wherein the amino group is protonated toform a salt (e.g., a hydrochloride salt), or wherein the amino group isfurther functionalized with a fluorophore (e.g., fluorescein) or apolypeptide (e.g., an -Asp-Asp-Asp peptide, which may be acetylated atthe terminus).

In some embodiments, the compound has formula (Ia):

wherein R³ and R⁴ are as defined in any of the embodiments describedabove for formula (I).

In some embodiments compound has formula (Ib):

wherein:

Y is selected from the group consisting of —NR^(a)R^(b) and —OR^(c);

R^(a) and R^(b) are each independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₈ alkyl, optionallysubstituted C₃-C₈-cycloalkyl, and optionally substituted heterocyclyl;or R^(a) and R^(b), together with the nitrogen atom to which they areattached, together form an optionally substituted ring; and

R^(c) is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl.

In some embodiments, Y is —OR^(c). In some embodiments, R^(c) isselected from the group consisting of hydrogen and methyl.

In some embodiments, Y is —NR^(a)R^(b).

In some embodiments, R^(a) is hydrogen. In some embodiments, R^(b) isselected from the group consisting of hydrogen, optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈-cycloalkyl, and optionallysubstituted heterocyclyl. In some embodiments, R^(b) is selected fromthe group consisting of hydrogen, unsubstituted C₁-C₆ alkyl (e.g.,n-butyl or n-hexyl), hydroxyalkyl (e.g., hydroxy-C₁-C₆-alkyl, such as(CH₂)₂OH or —(CH₂)₆OH), optionally substituted aminoalkyl (e.g.,amino-C₁-C₆-alkyl, such as —(CH₂)₆NH₂), dialkylaminoalkyl (e.g.,C₁-C₄-dialkylamino-C₁-C₈-alkylamido, such as —C(O)NH—(CH₂)₂N(CH₃)₂),carboxyalkyl (e.g., carboxy-C₁-C₈-alkyl, such as —(CH₂)₃COOH,—(CH₂)₅COOH or —(CH₂)₇COOH), sulfonic acid-C₁-C₈-alkyl (e.g.,—(CH₂)₆SO₃H), sulfonate-C₁-C₈-alkyl (e.g., —(CH₂)₆SO₃ ⁻),alkylcarbonylalkyl (e.g., C₁-C₄-alkylcarbonyl-C₁-C₈-alkylamido, such as—(CH₂)₃COOCH₃, —(CH₂)₅COOCH₃ or —(CH₂)₇COOCH₃), optionally substitutedC₃-C₆-cycloalkyl (e.g., -cyclohexyl, -cyclohexyl-COOH or-cyclohexyl-COOCH₃), and optionally substituted six-memberedheterocyclyl (e.g., unsubstituted or substituted with methyl,tert-butoxycarbonyl or acetyl). For example, in some embodiments, R^(b)is optionally substituted amino-C₁-C₈-alkyl (e.g., —(CH₂)₆NH₂), whereinthe amino group is protected with an amino protecting group (e.g.,tert-butoxycarbonyl), or wherein the amino group is protonated to form asalt (e.g., a hydrochloride salt), or wherein the amino group is furtherfunctionalized with a fluorophore (e.g., fluorescein) or a polypeptide(e.g., an -Asp-Asp-Asp peptide, which may be acetylated at theterminus).

In some embodiments R^(a) and R^(b), together with the nitrogen atom towhich they are attached, together form an optionally substituted ring.In some embodiments, R^(a) and R^(b), together with the nitrogen atom towhich they are attached, together form an optionally substitutedmonocyclic heterocycle. In some embodiments, the optionally substitutedmonocyclic heterocycle is optionally substituted piperidine. In someembodiments, the monocyclic heterocycle is selected from the groupconsisting of unsubstituted piperidine and piperidine substituted withone substituent (e.g., carboxyl or alkoxycarbonyl such as —C(O)OCH₂CH₃).

In some embodiments, the compound has the following formula (Ib′)

wherein Y is as defined in any of the embodiments described above forformula (Ib).

Suitable compounds include the following:

N-cyclohexyl-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-ethyl-2-(5-(pyrrolidine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide;

N-ethyl-2-(5-(piperidine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide;

ethyl1-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)piperidine-4-carboxylate;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-phenyl-4H-thieno[3,2-b]pyrrole-5-carboxamide;

ethyl2-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)phenyl)acetate;

methyl3-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)benzoate;

methyl-cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

8-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)octanoicacid;

6-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)piperidin-1-yl)-6-oxohexanoicacid;

trans-methyl-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid;

N-(trans-4-(butylcarbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((2-hydroxyethyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-(trans-4-((2-(dimethylamino)ethyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)butanoicacid;

N-(trans-4-carbamoylcyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-(hexylcarbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

ethyl1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carbonyl)piperidine-4-carboxylate;

methyl6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoate;

6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoicacid;

1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carbonyl)piperidine-4-carboxylicacid;

8-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)octanoicacid;

N-(trans-4-(cyclohexylcarbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((1-methylpiperidin-4-yl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

tert-butyl4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)piperidine-1-carboxylate;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-(piperidin-4-ylcarbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-(trans-4-((1-acetylpiperidin-4-yl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

tert-butyl(6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexyl)carbamate;

N-(trans-4-((6-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5(6)-carboxamido)hexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-(trans-4-((6-aminohexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamidehydrochloride;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

methyl-trans-4-(trans-4-(4-(2-(ethcyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)cyclohexane-1-carboxylate;

trans-4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)cyclohexane-1-carboxylicacid;

(11S,14S,17S)-17-acetamido-11,14-bis(carboxymethyl)-1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexyl)-1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecan-19-oicacid;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-methyl-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-cyclopentyl-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(pyridin-4-ylmethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(3-morpholinopropyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-ethyl-2-(5-(4-methylpiperazine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide;

methyl4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate;

N-cyclohexyl-4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-cyclohexyl-4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

methyl-trans-4-(4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyl4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate;

N-cyclohexyl-4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

methyl-trans-4-(4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyl4-(2-(benzyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate;

6-(cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoicacid;

methyl6-(trans-4-((4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)methyl)cyclohexane-1-carboxamido)hexanoate;

6-(trans-4-((4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)methyl)cyclohexane-1-carboxamido)hexanoicacid;

sodium6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;

potassium6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;

trans-4-(4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid;

methyltrans-4-(4-(2-(ethyl(phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-((3-cyanophenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-((3-carbamoylphenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(3-methoxyphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(o-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(6-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(p-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6-methoxy-1H-indole-2-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-furo[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

sodium6-(trans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;

methyltrans-4-(4-(2-(ethyl(3-isopropylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(3-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-((3-(bromomethyl)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-((3-(dimethylamino)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(-isobutylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate;

1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-1H-indole-2-carboxamide;

sodium6-(trans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;

methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-pyrrole-2-carboxamido)cyclohexane-1-carboxylate;and

methyltrans-4-(6-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6H-thieno[2,3-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate.

(1) Salt Forms

A thienopyrrole compound described herein can be in the form of a salt.A neutral form of the compound may be regenerated by contacting the saltwith a base or acid and isolating the parent compound in a conventionalmanner. The parent form of the compound differs from the various saltforms in certain physical properties, such as solubility in polarsolvents, but otherwise the salts are equivalent to the parent form ofthe compound for the purposes of this disclosure.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations. Examples ofsuitable organic cations include, but are not limited to, ammonium ion(i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R₁ ⁺, NH₂R₂ ⁺,NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions arethose derived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine.

If the compound is cationic, or has a functional group that may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucoheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Unless otherwise specified, a reference to a particular thienopyrrolecompound herein also includes salt forms thereof.

(2) Isomers

Certain thienopyrrole compounds may exist in one or more particulargeometric, optical, enantiomeric, diastereomeric, epimeric, atropic,stereoisomer, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; a- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referredto as “isomers” (or “isomeric forms”).

In some embodiments, a compound described herein may be anenantiomerically enriched isomer of a stereoisomer described herein. Forexample, the compound may have an enantiomeric excess of at least about10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Enantiomer, when used herein,refers to either of a pair of chemical compounds whose molecularstructures have a mirror-image relationship to each other.

In some embodiments, a preparation of a compound disclosed herein isenriched for an isomer of the compound having a selectedstereochemistry, e.g., R or S, corresponding to a selected stereocenter.For example, the compound has a purity corresponding to a compoundhaving a selected stereochemistry of a selected stereocenter of at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

In some embodiments, a composition described herein includes apreparation of a compound disclosed herein that is enriched for astructure or structures having a selected stereochemistry, e.g., R or S,at a selected stereocenter. Exemplary R/S configurations can be thoseprovided in an example described herein.

An “enriched preparation,” as used herein, is enriched for a selectedstereoconfiguration of one, two, three or more selected stereocenterswithin the subject compound. Exemplary selected stereocenters andexemplary stereoconfigurations thereof can be selected from thoseprovided herein, e.g., in an example described herein. By enriched ismeant at least 60%, e.g., of the molecules of compound in thepreparation have a selected stereochemistry of a selected stereocenter.In an embodiment it is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99%. Enriched refers to the level of a subject molecule(s)and does not connote a process limitation unless specified.

Thienopyrrole compounds may be prepared in racemic form or as individualenantiomers or diastereomers by either stereospecific synthesis or byresolution. The compounds may, for example, be resolved into theircomponent enantiomers or diastereomers by standard techniques, such asthe formation of stereoisomeric pairs by salt formation with anoptically active base, followed by fractional crystallization andregeneration of the free acid. The compounds may also be resolved byformation of stereoisomeric esters or amides, followed bychromatographic separation and removal of the chiral auxiliary.Alternatively, the compounds may be resolved using a chiral HPLC column.The enantiomers also may be obtained from kinetic resolution of theracemate of corresponding esters using lipase enzymes.

Except as discussed below for tautomeric forms, specifically excludedfrom the term “isomers,” as used herein, are structural (orconstitutional) isomers (i.e., isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g., C₃-alkyl or propylincludes n-propyl and iso-propyl; C₄-alkyl or butyl includes n-, iso-,sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, andpara-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol,amidine/amidine, nitroso/oxime, thioketone/enethiol,N-nitroso/hydroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

3. OPLOPHORUS LUCIFERASES

The thienopyrrole compounds of the present invention may be used toinhibit Oplophorus-derived luciferases. The thienopyrrole compounds mayinhibit the luciferase activity of the Oplophorus-derived luciferases.The Oplophorus-derived luciferase may be a wild-type Oplophorusluciferase or a variant of an Oplophorus luciferase, such as aluciferase of SEQ ID NO:2. Oplophorus luciferase variants are describedin U.S. Pat. Nos. 8,557,970 and 8,669,103, each of which is incorporatedherein by reference in its entirety.

The polypeptide sequence of the mature 19 kDa subunit of thenaturally-occurring form of the Oplophorus gracilirostris luciferase isprovided in SEQ ID NO: 1. An exemplary polypeptide sequence for asynthetic Oplophorus-derived luciferase, which can be used in themethods described herein, is provided in SEQ ID NO: 2 (alsointerchangeably referred to herein as “NanoLuc”, “Nluc,” “Nlucluciferase,” and “Nluc enzyme”).

4. COELENTERAZINE SUBSTRATES

The thienopyrrole compounds of the present invention may be used toinhibit luciferase activity by competing or interfering with acoelenterazine or coelenterazine-derivative substrate from binding to aluciferase. Coelenterazine substrates are a class of reporter moleculesthat luminesce when acted upon by luciferases and other bioluminescentproteins. Examples of coelenterazine substrates include but are notlimited to: coelenterazine; coelenterazine derivatives and/or analogssuch as 2-furanylmethyl-deoxy-coelenterazine (furimazine),coelenterazine-n, coelenterazine-f, coelenterazine-h,coelenterazine-hcp, coelenterazine-cp, coelenterazine-c,coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine(“coelenterazine-hh”), coelenterazine-i, coelenterazine-icp,coelenterazine-v, and 2-methyl-coelenterazine, in addition to thosedisclosed in WO 2003/040100, U.S. Patent Publication No. 2008/0248511,and U.S. Patent Publication No. US 2012/0117667; pro-coelenterazines(i.e. compounds that are not substrates for a non-luminescent enzyme,which converts the compound to a substrate for a luciferase),quinone-masked coelenterazines, and the like. Further examples ofcoelenterazine substrates are described in, for example, U.S.Publication No. 2012/0107849, U.S. Publication No. 2013/0130289, U.S.patent application Ser. No. 14/608,910, and U.S. patent application Ser.No. 14/609,372, each of which is incorporated herein by reference.

5. METHODS OF INHIBITING OPLOPHORUS LUCIFERASE ACTIVITY

The disclosed thienopyrrole compounds may be used in methods to inhibitOplophorus luciferase activity. The method may include contacting athienopyrrole compound disclosed herein (e.g., a compound of formula(I), (Ia), (Ib) or (Ib′) to a cell expressing or containing anOplophorus-derived luciferase, wherein the disclosed compounds mayselectively inhibit the Oplophorus-derived luciferase. The disclosedthienopyrrole compounds may be used in assays that are used detect thepresence or activity of enzymes using Oplophorus luciferases, toselectively inhibit the signal from the Oplophorus luciferase. Forexample, they may be used in a bioluminogenic method which employs anOplophorus luciferase and a coelenterazine or coelenterazine-derivativesubstrate to detect one or more molecules in a sample, e.g., a proteinof interest (e.g., an enzyme, a binding partner, a ligand, etc.), acofactor for an enzymatic reaction, an enzyme substrate, an enzymeinhibitor, an enzyme activator, or OH radicals, or one or moreconditions, e.g., redox conditions. While the coelenterazine substrateserves as a substrate for the Oplophorus luciferase, the claimedthienopyrrole compounds may serve to inhibit the luciferase toselectively suppress the luminescent signal in embodiments in which suchsuppression may be desired, such as in applications involving temporalmultiplexing of multiple bioluminescent systems, or in some plate-basedluminescent assays. For example, the thienopyrrole compounds may be usedto inhibit intracellular and/or extracellular Oplophorus luciferaseactivities.

(1) Thienopyrrole Compounds

As discussed above, the methods may include contacting a thienopyrrolecompound disclosed herein (e.g., a compound of formula (I), (Ia), (Ib)or (Ib′) to a cell expressing an Oplophorus-derived luciferase, whereinthe disclosed compounds may selectively inhibit the Oplophorus-derivedluciferase. In addition to compounds of formulae (I), (Ia), (Ib) and(Ib′), compounds that can be used in the methods described herein alsoinclude compounds of formula (II), or salts thereof:

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring.

In some embodiments, n is 1.

In some embodiments, the dashed line represents the presence of a bond,and X is CH.

In some embodiments, A is a phenyl ring. In some embodiments, A is a5-membered heteroaryl ring. In some embodiments, A is a thienyl ring. Insome embodiments, A is a furanyl ring.

In some embodiments, le is selected from the group consisting ofhydrogen, C₁-C₈ alkyl, halo-C₁-C₈-alkyl, alkoxyalkoxyalkyl andarylalkyl. For example, in some embodiments, R¹ is selected from thegroup consisting of hydrogen, ethyl, n-hexyl, 2-(2-methoxyethoxy)ethyland benzyl. In some embodiments, R¹ is ethyl.

In some embodiments, R² is optionally substituted aryl. For example, insome embodiments, R² is substituted phenyl. R² is phenyl substitutedwith one substituent selected from the group consisting of C₁-C₄ alkyl,cyano, amido, C₁-C₄ alkoxy, and hydroxyalkyl. In some embodiments, R² isphenyl substituted with one methyl group (e.g., m-tolyl).

In some embodiments, R¹ is C₁-C₄ alkyl and R² is optionally substitutedphenyl. In some embodiments, R¹ is ethyl and R² is m-tolyl.

In some embodiments, Z is —NR³R⁴.

In some embodiments, R³ and R⁴, together with the nitrogen atom to whichthey are attached, together form an optionally substituted ring. In someembodiments, R³ and R⁴, together with the nitrogen atom to which theyare attached, together form an optionally substituted monocyclicheterocycle. In some embodiments, the optionally substituted monocyclicheterocycle is selected from the group consisting of optionallysubstituted pyrrolidine, piperidine and piperazine. In some embodiments,the optionally substituted monocyclic heterocycle is selected from thegroup consisting of unsubstituted pyrrolidine, unsubstituted piperidine,piperidine substituted with one substituent (e.g., alkoxycarbonyl suchas ethoxycarbonyl), or piperazine substituted with one substituent(e.g., C₁-C₄ alkyl such as methyl).

In some embodiments, R³ is hydrogen.

In some embodiments, R⁴ is selected from the group consisting ofunsubstituted C₁-C₈ alkyl (e.g., methyl), carboxy-C₁-C₈-alkyl (e.g.,—(CH₂)₇—COOH), C₁-C₄-alkoxycarbonyl-C₁-C₈-alkyl (e.g., —(CH₂)₇—COOCH₃),optionally substituted phenyl (below), optionally substituted C₅-C₆cycloalkyl (e.g., unsubstituted cyclopentyl, unsubstituted cyclohexyl,or substituted cyclohexyl), optionally substituted heterocyclyl (e.g.,unsubstituted piperidinyl, piperidinyl substituted withtert-butoxycarbonyl, alkoxycarbonylalkylcarbonyl such as—CO—(CH₂)₄—COOCH₃), or carboxyalkylcarbonyl such as —CO—(CH₂)₄—COOH),optionally substituted heteroarylalkyl (e.g., pyridyl-C₁-C₄-alkyl suchas —CH₂-pyridyl), and optionally substituted heterocyclylalkyl (e.g.,morpholino-C₁-C₄-alkyl such as —(CH₂)₃-morpholino). For example, in someembodiments, R⁴ is phenyl that is unsubstituted or substituted with onesubstituent, such as a substituent selected from the group consisting ofC₁-C₄-alkoxycarbonyl (e.g., —C(O)OCH₃) andC₁-C₄-alkoxycarbonyl-C₁-C₄-alkyl (e.g., —CH₂—C(O)CH₂CH₃).

In some embodiments, R⁴ is cyclohexyl substituted with one substituentselected from the group consisting of carboxy, C₁-C₄-alkoxycarbonyl(e.g., —C(O)OCH₃), C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₃CH₃ or—C(O)NH—(CH₂)₅CH₃), hydroxy-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₂OH or—C(O)NH—(CH₂)₆OH), amido (i.e. -CONH₂), optionally substitutedamino-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₆NH₂),C₁-C₄-dialkylamino-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₂N(CH₃)₂),carboxy-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₃COOH, —C(O)NH—(CH₂)₅COOHor —C(O)NH—(CH₂)₇COOH), sulfonic acid-C₁-C₈-alkylamido (e.g.,—C(O)NH—(CH₂)₆SO₃H), sulfonate-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₆SO₃⁻), C₁-C₄-alkylcarbonyl-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₃COOCH₃,—C(O)NH—(CH₂)₅COOCH₃ or —C(O)NH—(CH₂)₇COOCH₃), optionally substitutedC₃-C₆-cycloalkylamido (e.g., —C(O)NH-cyclohexyl, —C(O)NH-cyclohexyl-COOHor —C(O)NH-cyclohexyl-COOCH₃), and heterocyclylamido (e.g.,—C(O)NH-piperidinyl, unsubstituted or substituted with methyl,tert-butoxycarbonyl or acetyl). For example, in some embodiments, R⁴ isoptionally substituted amino-C₁-C₈-alkylamido (e.g., —C(O)NH—(CH₂)₆NH₂),wherein the amino group is protected with an amino protecting group(e.g., tert-butoxycarbonyl), or wherein the amino group is protonated toform a salt (e.g., a hydrochloride salt), or wherein the amino group isfurther functionalized with a fluorophore (e.g., fluorescein) or apolypeptide (e.g., an -Asp-Asp-Asp peptide, which may be acetylated atthe terminus).

In some embodiments, Z is —OR⁵. In some embodiments, R⁵ is H. In someembodiments, R⁵ is optionally substituted C₁-C₄ alkyl (e.g., methyl).

(2) Use of Cell-Impermeable Thienopyrrole Compounds

In certain embodiments, the methods disclosed herein include contactinga sample (e.g., a cell) with a mixture of a cell-permeablecoelenterazine substrate and a compound described herein that ismodified such that it is cell-impermeable. In such embodiments, thedisclosed thienopyrrole compounds and methods may be used to build upthe initial brightness of a high-throughput screening operation assayformat, and then selectively inhibit any luciferases that may beexcreted from cells, to selectively inhibit luminescence that may occuroutside of the cells. Such methods may provide for a more selectivesignal within cells. Examples of cell-impermeable thienopyrrolecompounds include JRW-0051, JRW-0147, and JRW-0187.

(3) Use of Cell-Permeable Thienopyrrole Compounds

In certain embodiments, the methods disclosed herein include contactinga sample (e.g., a cell) with a mixture of a cell-permeablecoelenterazine substrate and a compound described herein that iscell-permeable. In such embodiments, the disclosed thienopyrrolecompounds can enter in to cells and selectively inhibit an Oplophorusluciferase therein. Such methods may be advantageous in multiplexingassays that involve use of two or more luciferases, and may allow forinhibition of luminescence from an Oplophorus luciferase so as toselectively view luminescence from another luciferase inside the cell.Examples of cell-permeable thienopyrrole compounds include JRW-0013 andJRW-0138.

(4) Use with Transcriptional Reporters

The disclosed thienopyrrole compounds may be used with genetictranscriptional reporter systems. In certain embodiments, provided is amethod for measuring the activity of a promoter in a sample, wherein thepromoter is operably linked to a gene encoding an Oplophorus-derivedluciferase or a variant thereof. The method includes (a) contacting thesample with a coelenterazine substrate; (b) determining the activity ofthe promoter by measuring luminescence of the sample, wherein the samplecomprises the promoter. The method can further include a step ofcontacting the sample with a thienopyrrole compound described herein, toselectively inhibit the luminescence. The promoter may be operablylinked to the gene via a translational or transcriptional fusion. Abiological pathway of interest, for example, may be examined by treatinga cell that comprises the promoter, which is operably linked to a geneencoding the luciferase, with an inducer agent of the pathway. Thispromoter activity may then be measured and monitored to study anycorrelation between the activity of the promoter and the pathway ofinterest, as well as obtain kinetic measurements relating to geneexpression (e.g. inducibility, repression and activation). Thethienopyrrole compound described herein can be used to selectivelyinhibit the luminescence.

(5) Multiplexing

The disclosed thienopyrrole compounds may be used to inhibit Oplophorusluciferases as applied to temporal multiplexing with other luciferasesand assays. In some embodiments, the Oplophorus-derived luciferase orvariant thereof may be multiplexed with another enzyme (e.g. aluciferase) that emits light at a different wavelength, e.g., greenfirefly luciferase, e.g., Photinus pyralis (e.g., Luc2; Promega Corp) orred click beetle luciferase (CHROMA-LUC™ luciferase; Promega Corp.). Forexample, if an Oplophorus luciferase is used as a functional reporter,then the green firefly luciferase or red CHROMA-LUC™ luciferase could beused to control for non-specific effects on genetic regulation or tonormalize for transfection efficiency. In some embodiments, luminescencegenerated from the Oplophorus luciferase (approximately 460 nm) and redCHROMA-LUC (approximately 610 nm) can be easily resolved using aluminometer with wavelength-discriminating filters, enabling themeasurement of both signals from the same sample. In such embodiments, athienopyrrole compound described herein can be used to selectivelyinhibit the Oplophorus luciferase, such that the signal from the otherluciferase can be selectively viewed.

In another example, an Oplophorus luciferase could be used as atranscriptional reporter and paired with a luciferase that emits lightat a different wavelength contained in an assay reagent. In anotherexample, an Oplophorus luciferase may be used with one or moreadditional luciferases, where the luminescence of each luciferase may beseparately measured through the use of selective enzyme inhibitors. Forexample, the luminescence of the Oplophorus luciferase may be measuredupon addition of appropriate substrates and buffers, followed bymeasurement of a second luciferase upon a subsequent addition ofappropriate substrates and buffers and one or more thienopyrrolecompounds described herein, which are selective for the an Oplophorusluciferase. In another example, the Oplophorus luciferase contained inan assay reagent may be used for measuring a specific aspect of cellularphysiology, for example ATP to estimate cell viability or caspaseactivity to estimate cellular apoptosis.

(6) Bioluminescence Resonance Energy Transfer (BRET)

The disclosed thienopyrrole compounds may be used in any method in whichan Oplophorus luciferase is used for detecting ligand-protein and/orprotein-protein interactions. In various embodiments, the Oplophorusluciferase may be used to transfer energy to an energy acceptor. Onesuch method is Bioluminescence Resonance Energy Transfer (BRET). Withrespect to BRET, energy transfer from a bioluminescent donor to afluorescent acceptor results in a shift in the spectral distribution ofthe emission of light. This energy transfer may enable real-timemonitoring of protein-protein or ligand-protein interaction in vitro orin vivo. In some embodiments, the BRET method may be an Nluc-MediatedBioluminescence Resonance Energy Transfer (such as NanoBRET) Assay forligand-protein and protein-protein interactions. NANOBRET comprises twodifferent methods: 1) using HALOTAG and Nluc-based technologies,Bioluminescence Resonance Energy Transfer (BRET) to detectprotein-protein and/or ligand-protein interactions may be achieved withincreased signal and decreased spectral overlap; and 2) using Nlucluciferase fused to a protein of interest and a fluorescent tracer todetect ligand-receptor interaction in living cells.

In some embodiments, the luminescent enzymes (i.e. Oplophorusluciferases) used in BRET analysis can be used to determine if twomolecules are capable of binding to each other or co-localize in a cell.For example, a luminescent enzyme can be used as a bioluminescence donormolecule, which is combined with a molecule or protein of interest tocreate a first fusion protein. In various embodiments, the first fusionprotein contains a luminescent enzyme and a protein of interest. Invarious embodiments, the first fusion proteins containing theluminescent enzyme can be used in BRET analysis to detectprotein/protein interaction in systems including but not limited to celllysates, intact cells, and living animals. In various embodiments,HALOTAG can be used as a fluorescent acceptor molecule. In someembodiments, HALOTAG can be fused to a second protein of interest or toa luminescent enzyme. For example, a luminescent enzyme can be fused toHALOTAG, expressed in cells or animals, and labeled with a fluorescentHALOTAG ligand such as HALOTAG TMR ligand. The fusion can subsequentlybe excited to fluoresce in the presence of a cell-permeant luminescentenzyme substrate. In some embodiments, BRET may be performed usingluminescent enzymes in combination with fluorescent proteins, includingbut not limited to Green Fluorescent Protein (GFP) or Red FluorescentProtein (RFP) or fluorescent labels including fluorescein, rhodaminegreen, Oregon green, or Alexa 488, to name a few non-limiting examples.

In some embodiments, quenching the signal from excreted Nluc can improvethe signal to noise ratio when using the NanoBRET plate assay.

In certain embodiments, a cell-permeable thienopyrrole compound may beused to inhibit intracellular BRET. In certain embodiments, acell-impermeable thienopyrrole compound may be used to inhibitextracellular BRET. In certain embodiments, a cell-impermeablethienopyrrole compound may be used in a target engagement model.

(7) Protein Proximity Assays for Live Cells or Lytic Formats

In some embodiments, Oplophorus luciferases may be used in circularlypermuted (CP) or straight split (SS) luminescent enzyme fusion proteinsto measure protein proximity. The Oplophorus luciferase is permuted orsplit via insertion of a protease substrate amino acid sequence (e.g.,TEV) to generate low bioluminescence. The inactive luciferase istethered (e.g., via genetic fusion) to a monitor protein. A potentialinteracting protein is tethered (e.g., via genetic fusion) to a protease(e.g., TEV). When the two monitor proteins interact or are in sufficientproximity (e.g., via a constitutive interaction, a drug stimulus or apathway response), the luminescent enzyme is cleaved to generateincreased bioluminescence activity. The example may be applied tomeasurements of protein proximity in cells or in biochemical assays.

(8) Protein Complementation Assays

In some embodiments, the disclosed thienopyrrole compounds may be usedto inhibit an Oplophorus luciferase when such a luciferase is used inother methods for detecting ligand-protein and protein-proteininteractions or proximity, such as the protein complementation assay(PCA) or enzyme fragmentation assay. Protein complementation assays(PCA) provide a means to detect the interaction of two biomolecules,e.g., polypeptides. PCA utilizes two fragments of the same protein,e.g., enzyme, that when brought into close proximity with each other canreconstitute into a functional, active protein. In some embodiments, theNANOBIT® technology (Promega Corporation) may be used to detectmolecular proximity by virtue of the reconstitution of a luminescentenzyme via the binding interaction of enzyme components or subunits.NANOBIT utilizes a non-luminescent peptide (NLPep) and non-luminescentpolypeptide (NLPoly) derived from the Oplophorus luciferase variant,Nluc luciferase. The NLPep and NLPoly are fused to proteins of interest.If the proteins of interest interact, NLPep and NLPoly interact toreconstitute a full-length Oplophorus luciferase enzyme.

For example, a luminescent enzyme can be separated into two fragments ata site(s) tolerant to separation and each fragment of the separatedluminescent enzyme can be fused to one of a pair of polypeptides ofinterest believed to interact, e.g., FKBP and FRB. If the twopolypeptides of interest do in fact interact, the luminescent enzymefragments, for example, then come into close proximity with each otherto reconstitute the functional, active luminescent enzyme. In someembodiments, the activity of the reconstituted luminescent enzyme canthen be detected and measured. In some embodiments, the splitluminescent enzyme can be used in a more general complementation systemsimilar to lac-Z (Langley et al., PNAS 72:1254-1257 (1975)) orribonuclease S (Levit and Berger, J. Biol. Chem. 251:1333-1339 (1976)).In some embodiments, a luminescent enzyme fragment (designated “A”)known to complement with another luminescent enzyme fragment (“B”) canbe fused to a target protein, and the resulting fusion can be monitoredvia luminescence in a cell or cell lysate containing fragment B. In someembodiments, the source of fragment B could be the same cell (e.g., ifthe gene for fragment B is integrated into the genome of the cell or iscontained on another plasmid within the cell) or it could be a lysate orpurified protein derived from another cell. In some embodiments, thissame fusion protein (fragment A) could be captured or immobilized usinga fusion between fragment B and a polypeptide such as HALOTAG capable ofattachment to a solid support. In some embodiments, luminescence can beused to demonstrate successful capture or to quantify the amount ofmaterial captured.

(9) Dimerization Assay

In some embodiments, the disclosed thienopyrrole compounds may be usedwith full-length circularly permuted luminescent enzymes fused torespective binding partners, e.g., FRB and FKBP, and used in a proteincomplementation-type assay. The key difference between the methoddisclosed herein and traditional protein complementation is that therewas no complementation, but rather there was dimerization of two fulllength enzymes, e.g., circularly permuted luminescent enzymes.

Briefly, the circularly permuted reporter proteins similarly configuredfor low activity are fused to both of the fusion protein partners. Forexample, each fusion partner may be linked to identically structured,permuted reporters. Interaction of the fusion partners brought thepermuted reporters into close proximity, thereby allowing reconstitutionof a hybrid reporter having higher activity.

6. SAMPLE

The disclosed thienopyrrole compounds may be used with samplescontaining biological components. The sample may comprise cells. Thesample may comprise heterogeneous mixtures of components (includingintact cells, cell extracts, cell lysates, bacteria, viruses,organelles, exosomes, and mixtures thereof) or a single component orhomogeneous group of components (e.g., natural or synthetic amino acid,nucleic acid or carbohydrate polymers, or lipid membrane complexes). Thethienopyrrole compounds may be generally non-toxic to living cells andother biological components within the concentrations of use.

The sample may include an animal (e.g., a vertebrate), a plant, afungus, physiological fluid (e.g., blood, plasma, urine, mucoussecretions and the like), a cell, a cell lysate, a cell supernatant, ora purified fraction of a cell (e.g., a subcellular fraction). In certainembodiments, the sample may be a cell. In some embodiments, the samplemay be a live cell. The cell may be a eukaryotic cell, e.g., yeast,avian, plant, insect or mammalian cells, including but not limited tohuman, simian, murine, canine, bovine, equine, feline, ovine, caprine orswine cells, or prokaryotic cells, or cells from two or more differentorganisms, or cell lysates or supernatants thereof. The cells may nothave been genetically modified via recombinant techniques(nonrecombinant cells), or may be recombinant cells which aretransiently transfected with recombinant DNA and/or the genome of whichis stably augmented with a recombinant DNA, or which genome has beenmodified to disrupt a gene, e.g., disrupt a promoter, intron or openreading frame, or replace one DNA fragment with another. The recombinantDNA or replacement DNA fragment may encode a molecule to be detected bythe methods of the invention, a moiety which alters the level oractivity of the molecule to be detected, and/or a gene product unrelatedto the molecule or moiety that alters the level or activity of themolecule. The cell may or may not express a luciferase. The cells mayhave been genetically modified via recombinant techniques.

7. KITS

Disclosed are kits for determining the presence or activity of one ormore enzymes (e.g., an Oplophorus or Oplophorus variant luciferase). Thekit may include one or more of the following: a compound or compositionof the invention that may inhibit the Oplophorus or Oplophorus variantluciferase, a coelenterazine or coelenterazine-derivative substrate, anOplophorus or Oplophorus variant luciferase, instructions for carryingout a luminescence assay, and reaction buffer(s). The reaction buffersmay be present in individual formulations for the non-luciferase enzymereactions and the luminescent enzyme reactions or in a singleformulation for a single step assay. The kits may also contain otherinhibitors, activators and/or enhancers for the non-luciferaseenzyme(s). The kits may also contain a positive and/or negative controlfor the assay.

8. EXAMPLES

General Synthesis Procedure A: To a solution of ester intermediate (1eq)in dioxane/water (4:1), lithium hydroxide (5eq) was added. Thesuspension was heated to 60° C. until starting material was consumed(monitored by LCMS or TLC analysis). The reaction mixture was cooled andacidified with HCl (2M) until pH 3. The suspension was partitionedbetween ethyl acetate and water. The organic layer was dried overanhydrous sodium sulfate and filtered. The filtrate was thenconcentrated. The residue was purified by column chromatography (silica,dichloromethane/methanol) to afford the desired product.

General Synthesis Procedure B: To a solution of carboxylate intermediate(1eq) in dimethylformamide, the requisite amine (or aminehydrochloride), hydroxybenzotriazole (2eq),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2eq) anddiisopropylethylamine (3eq) was added. The mixture was heated to 60° C.until starting material was consumed (monitored by LCMS or TLCanalysis). The reaction mixture cooled, diluted with ethyl acetate, andwashed with water. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was then concentrated. The residuewas purified by column chromatography (silica, dichloromethane/methanolor heptane/ethyl acetate) to afford the desired product.

Example 1 General Syntheses of Compounds of Formula (Ia)

Compounds of formula (Ia) can be generally synthesized according toScheme 1.

2-chloro-N-ethyl-N-(m-tolyl)acetamide (JRW-0003)

To a solution of N-ethyl-3-methylaniline (2.0 g, 14.8 mmol) in ethylacetate (25 mL), water (12 mL) was added. The biphasic solution wascooled to 0° C., and potassium hydroxide (2.49 g, 44.4 mmol) added inone motion. 2-Chloroacetyl chloride (2.5 g, 1.8 mL, 22.2 mmol) was addeddropwise over 10 min. The mixture was stirred for 1 h, diluted withwater, and extracted with ethyl acetate. The organic layer was driedover anhydrous sodium sulfate and filtered. The filtrate was thenconcentrated to afford crude product (3.2 g) as a mobile oil. ESI MS m/z212 [M+H]⁺.

methyl4-(2-(ethyl)m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0004)

To a solution of 2-chloro-N-ethyl-N-(m-tolyl)acetamide (14.8 mmol) inacetonitrile (100 mL), methyl 4H-thieno[3,2-b]pyrrole-5-carboxylate(2.28 g, 12.6 mmol), potassium carbonate (2.09 g, 15.1 mmol) and18-crown-6 (166 mg, 0.63 mmol) was added. The mixture was heated toreflux for 5 h, and the reaction was concentrated under vacuum to ˜20 mLvolume. The suspension was diluted with water, filtered, and washed withwater. The solid was dried under vacuum to afford crude product (4.6 g)as a light brown solid. ¹H NMR (300 MHz, DMSO-d₆) δ 7.53 (d, J=5.4, 1H),7.46-7.36 (m, 1H), 7.35-7.11 (m, 5H), 4.92 (s, 2H), 3.74 (s, 3H),3.67-3.53 (m, 2H), 2.37 (s, 3H), 1.00 (t, J=6.6, 3H); ESI MS m/z 357[M+H]⁺.

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (WZ-141-74)

Following general procedure A, methyl4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(2.0 g, 5.6 mmol) was reacted with lithium hydroxide (671 mg 28.0 mmol)to afford the desired product (1.8 g, 93%) as a light yellow solid. ¹HNMR (300 MHz, DMSO-d₆) δ 12.45 (s, 1H), 7.49 (d, J=5.4, 1H), 7.44-7.37(m, 1H), 7.33-7.12 (m, 4H), 7.09 (s, 1H), 4.91 (s, 2H), 3.71-3.54 (m,2H), 2.36 (s, 3H), 1.08-0.94 (s, 3H); ESI MS m/z 343 [M+H]⁺.

Example 2N-cyclohexyl-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0006)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (75 mg, 0.22 mmol) was reacted with cyclohexylamine (43 mg 0.44mmol) to afford the desired product (50 mg, 54%) as a white solid. ¹HNMR (300 MHz, DMSO-d₆) δ 7.83 (d, J=8.1, 1H), 7.44-7.16 (m, 5H),7.13-7.06 (m, 2H), 4.96 (s, 2H), 3.75-3.52 (m, 2H), 2.36 (s, 3H),1.84-1.51 (m, 5H), 1.37-1.17 (m, 6H), 1.05-0.93 (m, 3H); ESI MS m/z 424[M+H]⁺; HPLC>99% (AUC), T_(R) 7.02 min; UV (MeOH) λ_(max) 289 nm, ε25,200.

Example 3N-ethyl-2-(5-(pyrrolidine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide(JRW-0008)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (75 mg, 0.22 mmol) was reacted with pyrrolidine (43 mg 0.44 mmol)to afford the desired product (70 mg, 81%) as an off-white solid. ¹H NMR(300 MHz, DMSO-d₆) δ 7.45-7.16 (m, 5H), 7.06 (d, J=5.2, 1H), 6.83 (s,1H), 4.83 (s, 2H), 3.77-3.32 (m, 6H), 2.36 (s, 3H), 1.91-1.75 (m, 4H),1.06-0.92 (m, 3H); ESI MS m/z 396 [M+H]⁻; HPLC 97.3% (AUC), T_(R) 6.24min; UV (MeOH) λ_(max) 288 nm, ε 21,773.

Example 4N-ethyl-2-(5-(piperidine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide(JRW-0009)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (80 mg, 0.23 mmol) was reacted with piperidine (40 mg 0.47 mmol) toafford the desired product (90 mg, 94%) as an orange gum. ¹H NMR (300MHz, DMSO-d₆) δ 7.52-7.13 (m, 5H), 7.05 (d, J=5.2, 1H), 6.58 (s, 1H),4.75 (s, 2H), 3.68-3.45 (m, 6H), 2.37 (s, 3H), 1.69-1.42 (m, 6H), 1.00(t, J=6.9, 3H); ESI MS m/z 410 [M+H]⁺; HPLC 98.8% (AUC), T_(R) 5.91 min;UV (MeOH) λ_(max) 284 nm, ε 24,598.

Example 5 ethyl1-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)piperidine-4-carboxylate(JRW-0012)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (120 mg, 0.35 mmol) was reacted with ethyl piperidine-4-carboxylate(110 mg 0.70 mmol) to afford the desired product (160 mg, 94%) as awhite foam. ¹H NMR (300 MHz, DMSO-d₆) δ 7.47-7.36 (m, 1H), 7.32 (d,J=5.3, 1H), 7.29-7.14 (m, 3H), 7.07 (d, J=5.3, 1H), 6.61 (s, 1H), 4.75(s, 2H), 4.22 (d, J=13.2, 2H), 4.06 (q, J=7.1, 2H), 3.68-3.52 (m, 2H),3.18-2.94 (m, 2H), 2.70-2.55 (m, 1H), 2.37 (s, 3H), 1.90-1.78 (m, 2H),1.62-1.48 (m, 2H), 1.17 (t, J=7.1, 3H), 0.98 (t, J=6.8, 3H); ESI MS m/z482 [M+H]⁺; HPLC>99% (AUC), T_(R) 7.25 min; UV (MeOH) λ_(max) 286 nm, ε20,009

Example 64-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-phenyl-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0143)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (50 mg, 0.15 mmol) was reacted with aniline (16 mg 0.18 mmol) toafford the desired product (30 mg, 49%) as a foam. ¹H NMR (300 MHz,DMSO-d₆) δ 9.93 (s, 1H), 7.74-7.68 (m, 2H), 7.49-7.19 (m, 9H), 7.16 (d,J=5.3, 1H), 7.05 (t, J=7.4, 1H), 4.99 (s, 2H), 3.67-3.52 (m, 2H), 2.37(s, 3H), 1.06-0.92 (m, 3H); ESI MS m/z 418 [M+H]⁺; HPLC 85.4% (AUC),T_(R) 6.04 min; UV (EtOH) λ_(max) 306 nm, ε 27,330.

Example 7 ethyl2-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)phenyl)acetate(JRW-0152)

To a solution of4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (50 mg, 0.15 mmol) in DMF (3 mL), ethyl 2-(4-aminophenyl)acetate(31 mg, 0.18 mmol), HATU (111 mg, 0.29 mmol) and diisopropylethylamine(56 mg, 0.44 mmol) was added. The reaction was heated to 60° C. for 18h. The reaction mixture cooled, diluted with ethyl acetate, and washedwith water. The organic layer was dried over anhydrous sodium sulfateand filtered. The filtrate was then concentrated. The residue waspurified by column chromatography (silica, heptane/ethyl acetate) toafford the desired product (28 mg, 38%) as an orange solid. ¹H NMR (300MHz, DMSO-d₆) δ 9.93 (s, 1H), 7.64 (d, J=7.6, 2H), 7.50-7.11 (m, 9H),4.99 (s, 2H), 4.06 (q, J=6.4 Hz, 2H), 3.60 (s, 4H), 2.37 (s, 3H), 1.17(t, J=7.2, 3H), 0.98 (t, J=6.4, 3H); ESI MS m/z 476 [M+H]⁺; HPLC 97.2%(AUC), T_(R) 7.61 min; UV (EtOH) λ_(max) 308 nm, ε 34,350.

Example 8 methyl3-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)benzoate(JRW-0151)

To a solution of4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (50 mg, 0.15 mmol) in DMF (3 mL), methyl 3-aminobenzoate (33 mg,0.22 mmol), HATU (111 mg, 0.29 mmol) and diisopropylethylamine (56 mg,0.44 mmol). The reaction was heated to 60° C. for 18 h. The reactionmixture cooled, diluted with ethyl acetate, and washed with water. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was then concentrated. The residue was purified by columnchromatography (silica, heptane/ethyl acetate) to afford the desiredproduct (30 mg, 43%) as a light yellow solid. ¹H NMR (300 MHz, DMSO-d₆)δ 10.15 (s, 1H), 8.42-8.38 (m, 1H), 8.00 (d, J=8.2, 1H), 7.67-7.63 (m,1H), 7.56-7.10 (m, 8H), 5.00 (s, 2H), 3.86 (s, 3H), 3.67-3.55 (m, 2H),2.38 (s, 3H), 0.99 (t, J=6.7, 3H); ESI MS m/z 476 [M+H]⁺; HPLC 98.3%(AUC), T_(R) 7.52 min; UV (EtOH) λ_(max) 309 nm, ε 37,302.

Example 9methyl-cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0041)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (150 mg, 0.44 mmol) was reacted with methylcis-4-aminocyclohexane-1-carboxylate hydrochloride (127 mg, 0.66 mmol)to afford the desired product (186 mg, 88%) as a white foam. ¹H NMR (300MHz, DMSO-d₆) δ 7.82 (d, J=7.8, 1H), 7.45-7.33 (m, 2H), 7.32-7.16 (m,3H), 7.12 (s, 1H), 7.08 (d, J=5.3, 1H), 4.95 (s, 2H), 3.85-3.70 (m, 1H),3.68-3.53 (m, 5H), 2.63-2.56 (m, 1H), 2.36 (s, 3H), 2.08-1.92 (m, 2H),1.69-1.43 (m, 6H), 1.08-0.95 (m, 3H); ESI MS m/z 482 [M+H]⁺; HPLC>99%(AUC), T_(R) 7.16 min; UV (MeOH) λ_(max) 288 nm, ε 24,998.

Example 106-(cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoicacid (JRW-0264)

Step 1.cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (JRW-0261)

Following general procedure A,methyl-cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(160 mg, 0.33 mmol) was reacted with lithium hydroxide (40 mg, 1.66mmol) to afford crude product as a light brown solid. ESI MS m/z 468[M+H]⁺.

Step 2. methyl6-(cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoate(JRW-0262)

Following general procedure B,cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (68 mg, 0.15 mmol) was reacted with methyl 6-aminohexanoatehydrochloride (40 mg, 0.22 mmol) to afford the desired product (63 mg,72%) as a light red foam. ESI MS m/z 595 [M+H]⁺.

Step 3.6-(cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoicacid (JRW-0264)

Following general procedure A, methyl6-(cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoate(60 mg, 0.10 mmol) was reacted with lithium hydroxide (12 mg, 0.50 mmol)to afford the desired product (56 mg, 95%) as a light red foam. ¹H NMR(300 MHz, DMSO-d₆) δ 11.96 (s, 1H), 7.77 (d, J=7.2, 1H), 7.61 (t, J=5.5,1H), 7.45-7.33 (m, 2H), 7.33-7.14 (m, 4H), 7.08 (d, J=5.5, 1H), 4.95 (s,2H), 3.92-3.74 (m, 1H), 3.67-3.56 (m, 2H), 3.01 (dd, J=6.8, 12.6, 2H),2.36 (s, 3H), 2.27-2.10 (m, 3H), 1.94-1.79 (m, 2H), 1.78-1.64 (m, 2H),1.60-1.31 (m, 8H), 1.30-1.18 (m, 2H), 1.06-0.94 (m, 3H).; ESI MS m/z 581[M+H]⁺; HPLC 97.8% (AUC), T_(R) 5.50 min; UV (EtOH) λ_(max) 289 nm, ε18,873.

Example 118-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)octanoicacid (JRW-0198)

Step 1. methyl8-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)octanoate(JRW-0196)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (50 mg, 0.15 mmol) was reacted with methyl 8-aminooctanoatehydrochloride (46 mg, 0.22 mmol) to afford the desired product (72 mg,99%) as an oil. ESI MS m/z 498 [M+H]⁺.

Step 2.8-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)octanoicacid (JRW-0198)

Following general procedure A, methyl8-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)octanoate(72 mg, 0.14 mmol) was reacted with lithium hydroxide (17 mg, 0.72 mmol)to afford the desired product (47 mg, 67%) as a white foam. ¹H NMR (300MHz, DMSO-d₆) δ 11.91 (s, 1H), 8.09 (t, J=5.3, 1H), 7.47-7.16 (m, 5H),7.09 (d, J=5.3, 1H), 7.03 (s, 1H), 4.96 (s, 2H), 3.72-3.52 (m, 2H), 3.16(dd, J=6.4, 12.8, 2H), 2.36 (s, 3H), 2.17 (t, J=7.3, 2H), 1.60-1.40 (m,4H), 1.36-1.18 (m, 6H), 1.08-0.93 (m, 3H); ESI MS m/z 484 [M+H]⁺; HPLC99.4% (AUC), T_(R) 5.43 min; UV (EtOH) λ_(max) 288 nm, ε 24,627.

Example 126-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)piperidin-1-yl)-6-oxohexanoicacid (JRW-0208)

Step 1. tert-butyl4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)piperidine-1-carboxylate(JRW-0203)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (75 mg, 0.22 mmol) was reacted with tert-butyl4-aminopiperidine-1-carboxylate (66 mg, 0.33 mmol) to afford the desiredproduct (120 mg, quant) as a white foam. ESI MS m/z 525 [M+H]⁺.

Step 2.4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(piperidin-4-yl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0204)

To a solution of tert-butyl4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)piperidine-1-carboxylate(120 mg, 0.22 mmol) in dichloromethane (5 mL), trifluoroacetic acid (1mL) was added. The reaction stirred at RT for 1 h. The mixture wasdiluted with toluene and concentrated under vacuum (3×) to afford crudeproduct (170 mg) of light brown oil. ESI MS m/z 425 [M+H]⁺.

Step 3. methyl6-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)piperidin-1-yl)-6-oxohexanoate(JRW-0206)

To a solution of4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(piperidin-4-yl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(0.22 mmol) in dichloromethane (5 mL) cooled to 0° C., methyl6-chloro-6-oxohexanoate (49 mg, 0.27 mmol) and diisopropylethylamine(147 mg, 1.1 mmol) was added. The reaction stirred at 0° C. for 30 min.The mixture was diluted with dichloromethane and water, and the layerswere separated. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated. The residue waspurified by column chromatography (silica, dichloromethane/methanol) toafford the desired product (100 mg, 77%) as a white foam. ESI MS m/z 567[M+H]⁺.

Step 4.6-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)piperidin-1-yl)-6-oxohexanoicacid (JRW-0208)

Following general procedure A, methyl6-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)piperidin-1-yl)-6-oxohexanoate(100 mg, 0.18 mmol) was reacted with lithium hydroxide (21 mg 0.88 mmol)to afford the desired product (98 mg, quant.) as a white foam. ¹H NMR(300 MHz, DMSO-d₆) δ 11.96 (s, 1H), 7.93 (d, J=7.9, 1H), 7.46-7.34 (m,2H), 7.33-7.17 (m, 3H), 7.12-7.06 (m, 2H), 4.96 (s, 2H), 4.39-4.26 (m,1H), 4.05-3.75 (m, 2H), 3.68-3.54 (m, 2H), 3.17-3.00 (m, 1H), 2.73-2.57(m, 1H), 2.42-2.25 (m, 5H), 2.24-2.12 (m, 2H), 1.86-1.67 (m, 2H),1.55-1.11 (m, 6H), 1.07-0.93 (m, 3H); ESI MS m/z 553 [M+H]⁺; HPLC 98.8%(AUC), T_(R) 5.30 min; UV (MeOH) λ_(max) 289 nm, ε 23,015.

Example 13 methyl6-(trans-4-((4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)methyl)cyclohexane-1-carboxamido)hexanoate(JRW-0267)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (133 mg, 0.39 mmol) was reacted with methyl6-(trans-4-(aminomethyl)cyclohexane-1-carboxamido)hexanoate (111 mg,0.39 mmol) to afford the desired product (180 mg, 76%) as a white foam.¹H NMR (300 MHz, DMSO-d₆) δ 8.09 (t, J=5.6, 1H), 7.61 (t, J=5.5, 1H),7.66-7.56 (m, 2H), 7.32-7.16 (m, 3H), 7.14-6.99 (m, 2H), 4.96 (s, 2H),3.67-3.46 (m, 5H), 3.07-2.92 (m, 4H), 2.37 (s, 3H), 2.25 (t, J=7.4, 2H),2.08-1.93 (m, 1H), 1.83-1.60 (m, 4H), 1.56-1.12 (m, 10H), 1.08-0.80 (m,5H); ESI MS m/z 609 [M+H]⁺; HPLC>99% (AUC), T_(R) 5.95 min; UV (EtOH)λ_(max) 288 nm, ε 20,078.

Example 146-(trans-4-((4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)methyl)cyclohexane-1-carboxamido)hexanoicacid (JRW-0268)

Following general procedure A, methyl6-(trans-4-((4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)methyl)cyclohexane-1-carboxamido)hexanoate(165 mg, 0.27 mmol) was reacted with lithium hydroxide (32 mg 3.4 mmol)to afford the desired product (115 mg, 71%) as a white foam. ¹H NMR (300MHz, DMSO-d₆) δ 11.93 (s, 1H), 8.14-8.04 (m, 1H), 7.61 (t, J=5.3, 1H),7.46-7.16 (m, 5H), 7.13-6.98 (m, 2H), 4.96 (s, 2H), 3.68-3.53 (m, 2H),3.08-2.92 (m, 4H), 2.36 (s, 3H), 2.15 (t, J=7.2, 2H), 2.07-1.92 (m, 1H),1.80-1.63 (m, 4H), 1.54-1.14 (m, 10H), 1.08-0.79 (m, 5H); ESI MS m/z 595[M+H]⁺; HPLC>99% (AUC), T_(R) 5.44 min; UV (EtOH) λ_(max) 289 nm, ε25,604.

Example 15 General Synthesis of Compounds of Formula (Ib)

Compounds of formula (Ib) can be generally synthesized according toScheme 2.

Example 16trans-methyl-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0013)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (100 mg, 0.29 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (84 mg 0.44 mmol) to afford thedesired product (103 mg, 73%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆)δ 7.89 (d, J=7.9, 1H), 7.46-7.36 (m, 2H), 7.34-7.15 (m, 3H), 7.12-7.04(m, 2H), 4.96 (s, 2H), 3.72-3.46 (s, 6H), 2.36 (s, 3H), 2.32-2.18 (m,1H), 2.03-1.77 (m, 4H), 1.52-1.20 (m, 4H), 1.06-0.95 (m, 3H); ESI MS m/z482 [M+H]⁺; HPLC 99.0% (AUC), T_(R) 7.75 min; UV (MeOH) λ_(max) 289 nm,ε 26,100.

Example 17trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (JRW-0034)

Following general procedure A,trans-methyl-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(55 mg, 0.11 mmol) was reacted with lithium hydroxide (14 mg, 0.57 mmol)to afford the desired product (50 mg, 93%) as a white solid. ¹H NMR (300MHz, DMSO-d₆) δ 11.99 (s, 1H), 7.92-7.84 (m, 1H), 7.44-7.19 (m, 5H),7.12-7.04 (m, 2H), 4.97 (s, 2H), 3.73-3.52 (m, 3H), 2.36 (s, 3H),2.22-2.06 (m, 1H), 1.98-1.76 (m, 4H), 1.47-1.21 (m, 4H), 1.08-0.93 (m,3H); ESI MS m/z 468 [M+H]⁺; HPLC 99.4% (AUC), T_(R) 5.81 min; UV (MeOH)λ_(max) 290 nm, ε 26,502.

Example 18N-(trans-4-(butylcarbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0042)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with butylamine (9 mg, 0.13 mmol) toafford the desired product (50 mg, 89%) as a white solid. ¹H NMR (300MHz, DMSO-d₆) δ 7.82 (d, J=8.1, 1H), 7.65-7.57 (m, 1H), 7.46-7.11 (m,5H), 7.06-6.98 (m, 2H), 4.91 (s, 2H), 3.66-3.45 (m, 3H), 2.96 (q, J=6.0Hz, 2H), 2.32 (s, 3H), 2.05-1.92 (m, 1H), 1.85-1.60 (m, 4H), 1.50-1.10(m, 9H), 1.02-0.90 (m, 3H), 0.80 (t, J=7.2, 3H); ESI MS m/z 523 [M+H]⁺;HPLC 99.6% (AUC), T_(R) 6.52 min; UV (MeOH) λ_(max) 288 nm, ε 28,364.

Example 194-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((2-hydroxyethyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0043)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with ethanolamine (7 mg, 0.13 mmol)to afford the desired product (48 mg, 88%) as a white foam. ¹H NMR (300MHz, DMSO-d₆) δ 7.86 (d, J=7.8, 1H), 7.69 (t, J=5.4, 1H), 7.45-7.18 (m,5H), 7.13-7.04 (m, 2H), 4.96 (s, 2H), 4.60 (t, J=5.5, 1H), 3.75-3.54 (m,3H), 3.36 (q, J=5.9, 2H), 3.08 (q, J=5.9, 2H), 2.37 (s, 3H), 2.14-2.00(m, 1H), 1.90-1.69 (m, 4H), 1.52-1.10 (m, 4H), 1.08-0.94 (m, 3H); ESI MSm/z 511 [M+H]⁺; HPLC 99.7% (AUC), T_(R) 6.52 min; UV (MeOH) λ_(max) 289nm, ε 24,966.

Example 20N-(trans-4-((2-(dimethylamino)ethyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0044)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with ethanolamine (18 mg, 0.21 mmol)to afford the desired product (45 mg, 78%) as a light red solid. ¹H NMR(300 MHz, DMSO-d₆) δ 7.87 (d, J=8.1, 1H), 7.62 (t, J=5.5, 1H), 7.45-7.35(m, 2H), 7.33-7.17 (m, 3H), 7.12-7.05 (m, 2H), 4.97 (s, 2H), 3.73-3.54(m, 3H), 3.10 (q, J=6.4, 2H), 2.37 (s, 3H), 2.24 (t, J=6.4, 2H),2.15-2.00 (m, 7H), 1.88-1.691.78 (m, 4H), 1.53-1.19 (m, 4H), 1.08-0.94(m, 3H); ESI MS m/z 538 [M+H]⁺; HPLC 99.4% (AUC), T_(R) 4.31 min; UV(MeOH) λ_(max) 289 nm, ε 29,980.

Example 214-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)butanoicacid (JRW-0051)

Step 1. methyl4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)butanoate(JRW-0050)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with methyl 4-aminobutanoate (15 mg,0.13 mmol) to afford crude product (80 mg) as a white glass. ESI MS m/z567 [M+H]⁺.

Step 2.4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)butanoicacid (JRW-0051)

Following general procedure A, methyl4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)butanoate(60 mg, 0.10 mmol) was reacted with lithium hydroxide (13 mg, 0.53 mmol)to afford the desired product (55 mg, 93%) as a white solid. ¹H NMR (300MHz, DMSO-d₆) δ 11.97 (s, 1H), 7.82 (d, J=7.7, 1H), 7.66 (t, J=5.4, 1H),7.40-7.29 (m, 2H), 7.27-7.13 (m, 3H), 7.07-7.00 (m, 2H), 4.92 (s, 2H),3.68-3.48 (m, 3H), 3.03-2.92 (m, 2H), 2.32 (s, 3H), 2.14 (t, J=7.3, 2H),2.06-1.93 (m, 1H), 1.85-1.64 (m, 4H), 1.62-1.47 (m, 2H), 1.47-1.15 (m,4H), 1.05-0.88 (m, 3H); ESI MS m/z 553 [M+H]⁺; HPLC>99% (AUC), T_(R)5.17 min; UV (MeOH) λ_(max) 289 nm, ε 24,710.

Example 22N-(trans-4-carbamoylcyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0052)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with ammonia (1.1 mL, 0.5M, 0.21mmol) to afford the desired product (45 mg, 90%) as a white solid. ¹HNMR (300 MHz, DMSO-d₆) δ 7.82 (d, J=8.2, 1H), 7.40-7.29 (m, 2H),7.28-7.08 (m, 4H), 7.06-6.98 (m, 2H), 6.61 (s, 1H), 4.91 (s, 2H),3.67-3.49 (m, 3H), 2.31 (s, 3H), 2.03-1.91 (m, 1H), 1.82-1.68 (m, 4H),1.47-1.14 (m, 4H), 1.03-0.88 (s, 3H); ESI MS m/z 467 [M+H]⁺; HPLC>99%(AUC), T_(R) 5.11 min; UV (MeOH) λ_(max) 289 nm, ε 25,213.

Example 234-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-(hexylcarbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0138)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with hexylamine (13 mg, 0.13 mmol)to afford the desired product (50 mg, 85%) as a white solid. ¹H NMR (300MHz, DMSO-d₆) δ 7.88 (d, J=8.1, 1H), 7.67 (t, J=5.6, 1H), 7.45-7.34 (m,2H), 7.33-7.18 (m, 3H), 7.12-7.04 (m, 2H), 4.96 (s, 2H), 3.72-3.52 (m,3H), 2.99 (dd, J=6.5, 12.6, 2H), 2.36 (s, 3H), 2.10-1.96 (m, 1H),1.88-1.68 (m, 4H), 1.52-1.16 (m, 12H), 1.08-0.92 (m, 3H), 0.88-0.78 (m,3H); ESI MS m/z 551 [M+H]⁺; HPLC 99.4% (AUC), T_(R) 6.54 min; UV (EtOH)λ_(max) 292 nm, ε 29,535.

Example 24 ethyl1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carbonyl)piperidine-4-carboxylate(JRW-0140)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with ethyl piperidine-4-carboxylate(20 mg, 0.13 mmol) to afford the desired product (60 mg, 92%) as a clearsemi-solid. ¹H NMR (300 MHz, DMSO-d₆) δ 7.92 (d, J=7.8, 1H), 7.45-7.34(m, 2H), 7.32-7.18 (m, 3H), 7.12-7.05 (m, 2H), 4.96 (s, 2H), 4.23 (d,J=12.7, 1H), 4.05 (q, J=7.1, 2H), 3.87 (d, J=12.7, 1H), 3.72-3.52 (m,3H), 3.18-3.02 (m, 1H), 2.76-2.51 (m, 3H), 2.36 (s, 3H), 1.93-1.63 (m,6H), 1.56-1.26 (m, 6H), 1.16 (t, J=6.0 Hz, 3H), 1.07-0.93 (m, 3H); ESIMS m/z 607 [M+H]⁺; HPLC 99.5% (AUC), T_(R) 6.01 min; UV (EtOH) λ_(max)290 nm, ε 29,325.

Example 25 methyl6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoate(JRW-0145)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with methyl 6-aminohexanoate (23 mg,0.13 mmol) to afford the desired product (60 mg, 94%) as a white solid.¹H NMR (300 MHz, DMSO-d₆) δ 7.88 (d, J=8.1, 1H), 7.68 (t, J=5.6, 1H),7.44-7.34 (m, 2H), 7.32-7.17 (m, 3H), 7.12-7.03 (m, 2H), 4.95 (s, 2H),3.73-3.50 (m, 6H), 2.98 (dd, J=6.6, 12.5, 2H), 2.36 (s, 3H), 2.26 (t,J=7.4, 2H), 2.08-1.95 (m, 1H), 1.87-1.67 (m, 4H), 1.57-1.15 (m, 10H),1.05-0.93 (m, 3H); ESI MS m/z 595 [M+H]⁺; HPLC 99.4% (AUC), T_(R) 6.24min; UV (EtOH) λ_(max) 288 nm, ε 26,555.

Example 266-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoicacid (JRW-0147)

Following general procedure A, methyl6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoate(53 mg, 0.089 mmol) was reacted with lithium hydroxide (10 mg, 0.44mmol) to afford the desired product (50 mg, 96%) as a white solid. ¹HNMR (300 MHz, DMSO-d₆) δ 11.98 (s, 1H), 7.88 (d, J=7.6, 1H), 7.72-7.63(m, 1H), 7.44-7.33 (m, 2H), 7.32-7.18 (m, 3H), 7.12-7.04 (m, 2H), 4.95(s, 2H), 3.72-3.52 (m, 3H), 3.04-2.93 (m, 2H), 2.36 (s, 3H), 2.16 (t,J=7.1, 2H), 2.10-1.93 (m, 1H), 1.88-1.67 (m, 4H), 1.55-1.10 (m, 10H),1.08-0.92 (m, 3H); ESI MS m/z 581 [M+H]⁺; HPLC>99% (AUC), T_(R) 5.49min; UV (EtOH) λ_(max) 288 nm, ε 23,738.

Example 271-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carbonyl)piperidine-4-carboxylicacid (JRW-0187)

Following general procedure A, ethyl1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carbonyl)piperidine-4-carboxylate(60 mg, 0.099 mmol) was reacted with lithium hydroxide (12 mg, 0.49mmol) to afford the desired product (45 mg, 78%) as a white solid. ¹HNMR (300 MHz, DMSO-d₆) δ 12.20 (s, 1H), 7.90 (d, J=7.5, 1H), 7.45-7.33(m, 2H), 7.32-7.18 (m, 3H), 7.08 (s, 2H), 4.96 (s, 2H), 4.28-4.15 (m,1H), 3.92-3.79 (m, 1H), 3.70-3.52 (m, 3H), 3.15-3.00 (m, 1H), 2.75-2.60(m, 1H), 2.36 (s, 3H), 1.92-1.63 (m, 7H), 1.56-1.25 (m, 7H), 1.08-0.95(m, 3H); ESI MS m/z 579 [M+H]⁺; HPLC 99.5% (AUC), T_(R) 4.45 min; UV(EtOH) λ_(max) 289 nm, ε 23,470.

Example 288-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)octanoicacid (JRW-0188)

Step 1. methyl8-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)octanoate(JRW-0186)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with methyl 8-aminooctanoate (27 mg,0.13 mmol) to afford the desired product (55 mg, 82%) as an oil. ESI MSm/z 623 [M+H]⁺.

Step 2.8-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)octanoicacid (JRW-0188)

Following general procedure A, methyl8-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)octanoate(50 mg, 0.080 mmol) was reacted with lithium hydroxide (9 mg, 0.40 mmol)to afford the desired product (45 mg, 92%) as a white foam. ¹H NMR (300MHz, DMSO-d₆) δ 11.92 (s, 1H), 7.87 (d, J=7.8, 1H), 7.70-7.61 (m, 1H),7.45-7.14 (m, 5H), 7.10-7.03 (m, 2H), 4.96 (s, 2H), 3.72-3.54 (m, 3H),3.05-2.93 (m, 2H), 2.37 (s, 3H), 2.22-2.12 (m, 2H), 2.10-1.98 (m, 1H),1.91-1.66 (m, 4H), 1.55-1.10 (m, 14H), 1.08-0.93 (m, 3H); ESI MS m/z 609[M+H]⁺; HPLC 96.9% (AUC), T_(R) 4.97 min; UV (EtOH) λ_(max) 289 nm, ε25,824.

Example 29N-(trans-4-(cyclohexylcarbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0190)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with cyclohexylamine (16 mg, 0.16mmol) to afford the desired product (55 mg, 94%) as a white solid. ¹HNMR (300 MHz, DMSO-d₆) δ 7.95-7.82 (m, 1H), 7.66-7.16 (m, 6H), 7.12-7.04(m, 2H), 4.97 (s, 2H), 3.76-3.40 (m, 4H), 2.36 (s, 3H), 2.11-1.96 (m,1H), 1.90-1.60 (m, 8H), 1.59-0.91 (m, 13H); ESI MS m/z 549 [M+H]⁺; HPLC99.5% (AUC), T_(R) 5.98 min; UV (EtOH) λ_(max) 288 nm, ε 25,407.

Example 304-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((1-methylpiperidin-4-yl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0191)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (50 mg, 0.11 mmol) was reacted with 1-methylpiperidin-4-amine (18mg, 0.16 mmol) to afford the desired product (47 mg, 78%) as a whitesolid. ¹H NMR (300 MHz, DMSO-d₆) δ 7.87 (d, J=8.1, 1H), 7.58 (d, J=7.6,1H), 7.50-7.16 (m, 5H), 7.13-7.04 (m, 2H), 4.96 (s, 2H), 3.75-3.38 (m,4H), 2.73-2.61 (m, 2H), 2.36 (s, 3H), 2.22-1.96 (m, 5H), 1.96-1.58 (m,8H), 1.53-1.19 (m, 6H), 1.08-0.97 (s, 3H); ESI MS m/z 564 [M+H]⁺; HPLC98.8% (AUC), T_(R) 3.35 min; UV (EtOH) λ_(max) 289 nm, ε 30,051.

Example 31 tert-butyl4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)piperidine-1-carboxylate(JRW-0192)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (84 mg, 0.18 mmol) was reacted with tert-butyl4-aminopiperidine-1-carboxylate (54 mg, 0.27 mmol) to afford the desiredproduct (115 mg, 99%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ7.93-7.83 (m, 1H), 7.68-7.58 (m, 1H), 7.45-7.16 (m, 5H), 7.13-7.03 (m,2H), 4.96 (s, 2H), 3.96-3.46 (m, 6H), 2.93-2.70 (m, 2H), 2.36 (s, 3H),2.09-1.95 (m, 1H), 1.88-1.60 (m, 6H), 1.55-1.08 (m, 15H), 1.06-0.93 (m,3H); ESI MS m/z 650 [M+H]⁻; HPLC 99.6% (AUC), T_(R) 5.87 min; UV (EtOH)λ_(max) 288 nm, ε 31,128.

Example 324-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-(piperidin-4-ylcarbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0194)

To a solution of tert-butyl4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)piperidine-1-carboxylate(105 mg, 0.16 mmol) in dichloromethane (5 mL), trifluoroacetic acid (1mL) was added. The reaction was stirred at RT for 2 h. The mixture wasdiluted with toluene and concentrated under vacuum (3×) to afford crudeproduct (150 mg) of white foam. ESI MS m/z 550 [M+H]⁺.

Example 33N-(trans-4-((1-acetylpiperidin-4-yl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0195)

To a solution of4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-(piperidin-4-ylcarbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(0.16 mmol) in dichloromethane (5 mL) cooled to 0° C., acetyl chloride(25 mg, 0.32 mmol) and diisopropylethylamine (104 mg, 0.81 mmol) wasadded. The reaction stirred and warmed to RT overnight. The mixture wasdiluted with dichloromethane and water, and the layers were separated.The organic layer was dried over anhydrous sodium sulfate and filtered.The filtrate was then concentrated. The residue was purified by columnchromatography (silica, dichloromethane/methanol) to afford the desiredproduct (84 mg, 88%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 7.87(d, J=7.6, 1H), 7.67 (d, J=7.6, 1H), 7.46-7.32 (m, 2H), 7.33-7.17 (m,3H), 7.13-7.03 (m, 2H), 4.96 (s, 2H), 4.22-4.08 (m, 1H), 3.81-3.52 (m,5H), 3.15-3.00 (m, 1H), 2.70 (t, J=11.9, 1H), 2.36 (s, 3H), 2.11-1.94(m, 4H), 1.91-1.59 (m, 6H), 1.52-1.09(m, 6H), 1.06-0.91 (m, 3H); ESI MSm/z 592 [M+H]⁺; HPLC 99.8% (AUC), T_(R) 4.09 min; UV (EtOH) λ_(max) 288nm, ε 26,034.

Example 34 tert-butyl(6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexyl)carbamate(JRW-0197)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (115 mg, 0.25 mmol) was reacted with tert-butyl(6-aminohexyl)carbamate (80 mg, 0.37 mmol) to afford the desired product(150 mg, 91%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆) δ 7.87 (d,J=8.0, 1H), 7.65 (t, J=5.1, 1H), 7.45-7.33 (m, 2H), 7.32-7.17 (m, 3H),7.12-7.06 (m, 2H), 6.77-6.68 (m, 1H), 4.96 (s, 2H), 3.72-3.53 (m, 3H),3.03-2.93 (m, 2H), 2.87 (dd, J=6.0, 12.6, 2H), 2.36 (s, 3H), 2.12-1.96(m, 1H), 1.88-1.68 (m, 4H), 1.51-1.16 (m, 21H), 1.06-0.93 (m, 3H); ESIMS m/z 666 [M+H]⁺; HPLC 99.7% (AUC), T_(R) 4.11 min; UV (EtOH) λ_(max)288 nm, ε 21,608.

Example 35N-(trans-4-((6-(3′,6′-dihydroxy-3-oxo-3H-spirolisobenzofuran-1,9′-xanthene]-5(6)-carboxamido)hexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0200)

Step 1.N-(trans-4-((6-aminohexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0199)

To a solution of tert-butyl(6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexyl)carbamate(150 mg, 0.22 mmol) in dichloromethane (5 mL), trifluoroacetic acid (1mL) was added. The reaction stirred at RT for 5 h. The mixture wasdiluted with toluene and concentrated under vacuum (3×) to afford crudeproduct (150 mg) of clear oil. ESI MS m/z 566 [M+H]⁺.

Step 2.N-(trans-4-((6-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5(6)-carboxamido)hexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0200)

To a solution ofN-(trans-4-((6-aminohexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(0.22 mmol) in DMF (3 mL), 2,5-dioxopyrrolidin-1-yl3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5(6)-carboxylate[5(6)-FAM-SE] (106 mg, 0.22 mmol) and diisopropylethylamine (145 mg, 1.1mmol) was added. The reaction was stirred at RT for 1 h. The reactionmixture was acidified (0.1M HCl), diluted with water, and extracted with3:1 CHCl₃/isopropanol. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was then concentrated. The residuewas purified by column chromatography (silica, dichloromethane/methanol)to afford the desired product (145 mg, 69%) as an orange solid. ¹H NMR(300 MHz, DMSO-d₆) δ 10.12 (s, 2H), 8.78 (t, J=5.5, 1H, isomer A), 8.64(t, J=5.7, 1H, isomer B), 8.44 (s, 1H, isomer A), 8.23 (dd, J=1.5, 8.1,1H, isomer A), 8.15 (dd, J=1.3, 8.1, 1H, isomer B), 8.05 (d, J=8.1, 1H,isomer B), 7.87 (d, J=8.0, 1H), 7.72-7.60 (m, 1H), 7.44-7.32 (m, 3H),7.32-7.17 (m, 3H), 7.12-7.04 (m, 2H), 6.70-6.65 (m, 2H), 6.61-6.49 (m,4H), 4.96 (s, 2H), 3.70-3.52 (m, 3H), 3.32-3.23 (m, 2H), 3.22-3.12 (m,1H), 3.08-2.91 (m, 2H), 2.36 (s, 3H), 2.11-1.95 (m, 1H), 1.89-1.65 (m,4H), 1.61-1.16 (m, 11H), 1.06-0.92 (m, 3H); ESI MS m/z 925 [M+H]⁺; HPLC97.1% (AUC), T_(R) 6.16, 6.26 min; UV (MeOH) λ_(max) 284 nm, ε 31,419.

Example 36N-(trans-4-((6-aminohexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamidehydrochloride (JRW-0241)

To a solution of tert-butyl(6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexyl)carbamate(100 mg, 0.15 mmol) in dichloromethane (5 mL), trifluoroacetic acid (1mL) was added. The reaction stirred at RT for 1 h. The mixture wasdiluted with toluene and concentrated under vacuum (3×). The residue wasdissolved in methanol and HCl (2 mL, 1M in ether) was added. Thesolution was evaporated to afford the desired product (95 mg, quant.) ofwhite solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.02-7.68 (m, 5H), 7.45-7.34(m, 2H), 7.33-7.15 (m, 3H), 7.12-7.05 (m, 2H), 4.97 (s, 2H), 3.74-3.51(m, 3H), 3.01 (dd, J=6.5, 12.5, 2H), 2.79-2.66 (m, 2H), 2.36 (s, 3H),2.11-1.97 (m, 1H), 1.88-1.68 (m, 4H), 1.59-1.17 (m, 12H), 1.02-0.94 (m,3H); ESI MS m/z 566 [M+H]⁺; HPLC 99.0% (AUC), T_(R) 4.47 min; UV (MeOH)λ_(max) 289 nm, ε 23,213.

Example 374-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0242)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (150 mg, 0.32 mmol) was reacted with 6-aminohexan-1-ol (56 mg, 0.48mmol) to afford the desired product (160 mg, 88%) as a white foam. ¹HNMR (300 MHz, DMSO-d₆) δ 7.87 (d, J=7.4, 1H), 7.73-7.59 (s, 1H),7.48-7.15 (m, 5H), 7.13-7.02 (m, 2H), 4.96 (s, 2H), 4.35-4.23 (m, 1H),3.73-3.50 (m, 3H), 3.43-3.30 (m, 2H), 3.05-2.94 (m, 2H), 2.37 (s, 3H),2.10-1.95 (m, 1H), 1.90-1.65 (m, 4H), 1.55-1.13 (m, 12H), 1.09-0.90 (s,3H); ESI MS m/z 567 [M+H]⁺; HPLC>99% (AUC), T_(R) 5.48 min; UV (MeOH)λ_(max) 289 nm, ε 26,356.

Example 38 Sodium6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate(JRW-0344)

Step 1.4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((6-iodohexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0342)

To a solution of imidazole (36 mg, 0.53 mmol), triphenylphosphine (138mg, 0.53 mmol), and iodine (134 mg, 0.53 mmol),4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(100 mg, 0.18 mmol)dissolved in THF (5 mL) was added. The solutionstirred for 1 h at RT. The reaction was diluted with ethyl acetate andquenched with a 10% Na₂S₂O₃ solution. The mixture was diluted with ethylacetate and water, and the layers were separated. The organic layer waswashed with a 10% Na₂S₂O₃ solution and brine, dried over anhydroussodium sulfate, and filtered. The filtrate was then concentrated. Theresidue was partially purified by column chromatography (silica,dichloromethane/methanol) to afford crude product as a white solid. ESIMS m/z 677 [M+H]+.

Step 2. Sodium6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate(JRW-0344)

To a solution of4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((6-iodohexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(0.18 mmol) in ethanol (5 mL), sodium sulfite (66 mg, 0.53 mmol) andwater (3 mL)was added. The mixture was heated to 75° C. for 2 h. Thereaction was concentrated, and the residue was purified by columnchromatography (silica, dichloromethane/methanol) to afford the desiredproduct (100 mg, 90%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 7.87(d, J=8.0, 1H), 7.67 (t, J=5.5, 1H), 7.49-7.17 (m, 5H), 7.13-7.05 (m,2H), 4.97 (s, 2H), 3.72-3.54 (m, 3H), 3.05-2.93 (m, 2H), 2.40-2.28 (m,5H), 2.12-1.96 (m, 1H), 1.88-1.68 (m, 4H), 1.62-1.14 (m, 12H), 1.05-0.93(m, 3H); ESI MS m/z 631 [M+H]⁺; HPLC>99% (AUC), T_(R) 4.56 min; UV(MeOH) λ_(max) 288 nm, ε 21,072.

Example 39 Potassium6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate(JRW-0348)

To a solution of6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonicacid (50 mg, 0.08 mmol) in water (25 mL), DowEx 50WX4 (potassiumcharged) was added. The suspension stirred at RT for 10 min. The mixturewas filtered, and the filtrate was concentrated by lyophilization toafford the desired product (48 mg, 90%) as a white solid. ¹H NMR (300MHz, DMSO-d₆) δ 7.83 (d, J=8.1, 1H), 7.62 (t, J=5.5, 1H), 7.47-7.15 (m,5H), 7.10-7.04 (m, 2H), 4.97 (s, 2H), 3.71-3.54 (m, 3H), 3.05-2.94 (m,2H), 2.41-2.31 (m, 5H), 2.15-1.96 (m, 1H), 1.90-1.70 (m, 4H), 1.61-1.16(m, 12H), 1.06-0.95 (m, 3H); ESI MS m/z 631 [M+H]⁺; HPLC>99% (AUC),T_(R) 4.55 min; UV (MeOH) λ_(max) 289 nm, ε 19,812.

Example 40methyl-trans-4-(trans-4-(4-(2-(ethcyhm-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)cyclohexane-1-carboxylate(JRW-0243)

Following general procedure B,trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (75 mg, 0.16 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate hydrochloride (46 mg, 0.24 mmol)to afford the desired product (89 mg, 91%) as a white solid. ¹H NMR (300MHz, DMSO-d₆) δ 7.86 (d, J=8.5, 1H), 7.58 (d, J=7.8, 1H), 7.47-7.33 (m,2H), 7.32-7.18 (m, 3H), 7.11-7.04 (m, 2H), 4.96 (s, 2H), 3.72-3.53 (m,6H), 3.52-3.35 (m, 1H), 2.36 (s, 3H), 2.29-2.16 (m, 1H), 2.08-1.95 (m,1H), 1.95-1.65 (m, 8H), 1.53-1.06 (m, 8H), 1.05-0.93 (m, 3H); ESI MS m/z607 [M+H]⁺; HPLC 99.7% (AUC), T_(R) 6.27 min; UV (MeOH) λ_(max) 288 nm,ε 24,192.

Example 41trans-4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)cyclohexane-1-carboxylicacid (JRW-0245)

Following general procedure A,methyl-trans-4-(trans-4-(4-(2-(ethcyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)cyclohexane-1-carboxylate(80 mg, 0.13 mmol) was reacted with lithium hydroxide (16 mg, 0.66 mmol)to afford the desired product (66 mg, 84%) as a white solid. ¹H NMR (300MHz, DMSO-d₆) δ 12.00 (s, 1H), 7.86 (d, J=8.1, 1H), 7.57 (d, J=7.6, 1H),7.45-7.33 (m, 2H), 7.32-7.15 (m, 3H), 7.12-7.03 (m, 2H), 4.97 (s, 2H),3.73-3.52 (m, 3H), 3.52-3.35 (m, 1H), 2.36 (s, 3H), 2.19-1.95 (m, 2H),1.94-1.65 (m, 8H), 1.51-1.05 (m, 8H), 1.05-0.94 (m, 3H); ESI MS m/z 593[M+H]⁺; HPLC 98.4% (AUC), T_(R) 5.51 min; UV (MeOH) λ_(max) 288 nm, ε24,976.

Example 42(11S,14S,17S)-17-acetamido-11,14-bis(carboxymethyl)-1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexyl)-1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecan-19-oicacid (JRW-0251)

Step 1. tert-butyl(11S,14S,17S)-17-acetamido-11,14-bis(2-(tert-butoxy)-2-oxoethyl)-1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexyl)-1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecan-19-oate(JRW-0249)

Following general procedure B,(S)-2-((S)-2-((S)-2-acetamido-4-(tert-butoxy)-4-oxobutanamido)-4-(tert-butoxy)-4-oxobutanamido)-4-(tert-butoxy)-4-oxobutanoicacid (81 mg, 0.14 mmol) was reacted withN-(trans-4-((6-aminohexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamidehydrochloride (85 mg, 0.14 mmol) to afford the desired product (125 mg,78%) as a light yellow solid. ESI MS m/z 1122 [M+H]⁺.

Step 2.(11S,14S,17S)-17-acetamido-11,14-bis(carboxymethyl)-1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexyl)-1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecan-19-oicacid (JRW-0251)

To a solution of tert-butyl(11S,14S,17S)-17-acetamido-11,14-bis(2-(tert-butoxy)-2-oxoethyl)-1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexyl)-1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecan-19-oate(120 mg, 0.11 mmol) in dichloromethane (10 mL), trifluoroacetic acid (1mL) was added. The reaction stirred at RT for 18 h. The mixture wasdiluted with toluene and concentrated under vacuum (3×). The residue waspurified by column chromatography (silica, dichloromethane/methanol) toafford the desired product (68 mg, 66%) as a white solid. ¹H NMR (300MHz, DMSO-d₆) δ 12.31 (s, 3H), 8.45-8.18 (m, 2H), 7.95-7.83 (m, 2H),7.66 (t, J=5.5, 1H), 7.52-7.17 (m, 6H), 7.11-7.04 (m, 2H), 4.96 (s, 2H),4.55-4.35 (m, 3H), 3.71-3.52 (m, 2H), 3.06-2.90 (m, 4H), 2.75-2.60 (m,3H), 2.60-2.42 (m, 4H), 2.36 (s, 3H), 2.09-1.96 (m, 1H), 1.89-1.66 (m,7H), 1.53-1.13 (m, 12H), 1.06-0.93 (m, 3H); ESI MS m/z 953 [M+H]⁺; HPLC93.8% (AUC), T_(R) 4.74 min; UV (MeOH) λ_(max) 289 nm, ε 24,417.

Example 43 Alternate Syntheses of Compounds of Formula (Ia)

Compounds of formula (Ia) can also be generally synthesized according toScheme 3.

Example 444-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-methyl-4H-thieno[3,2-b]pyrrole-5-carboxamide(WZ-141-84)

Step 1. 2,5-dioxopyrrolidin-1-yl4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(WZ-141-82)

4-(2-(Ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (0.44 g, 1.28 mmol) and TSTU (1.16 g, 3.85 mol) was dissolved in 15ml of methylene chloride and 15 ml of acetonitrile. DIPEA (0.996 g, 7.71mmol) was slowly added at room temperature, and the resultant mixturewas stirred at room temperature for 30 minutes. The reaction mixture wasdiluted by 100 ml of methylene chloride, washed twice with 30% citricacid and twice with water, and dried over Na₂SO₄. The organic solventwas concentrated to 30 ml solution. Without further purification, aportion of the solution was used directly in next step.

Step 2.4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-methyl-4H-thieno[3,2-b]pyrrole-5-carboxamide(WZ-141-84)

To the above 10 ml of crude of 2,5-dioxopyrrolidin-1-yl4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(WZ141-82) (150 mg, 0.34 mmol), methyl amine (40%) (0.53 g, 6.83 mmol)was added, and the resulted mixture was stirred at room temperature for30 minutes. After removing solvent, the compound was purified by flashcolumn using heptane/ethyl acetate as eluent to give the desired productin a quantitative yield. ¹H NMR (300 MHz, CD₂Cl₂) δ 7.5-6.8 (m, 7H),4.95 (s, 2H), 3.76 (m, 2H), 2.91 (d, 3H), 2.43 (s, 3H), 1.10 (t, 3H);ESI MS m/z 356 [M+H]⁺; HPLC 99.6% at 254 nm.

Example 45N-cyclopentyl-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(WZ-141-88)

The compound WZ141-88 was synthesized by employing the similar methodfor preparation of4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-methyl-4H-thieno[3,2-b]pyrrole-5-carboxamide(WZ141-84). ¹H NMR (300 MHz, DMSO-d₆) δ 7.96 (d, 1H), 7.5-7.1 (m, 6H),6.8 (d, 1H), 4.90 (s, 2H), 4.61 (m, 1H), 3.63 (m, 2H), 2.38 (s, br, 3H),1.9-1.4 (m, 8H), 1.00 (t, 3H); ESI MS m/z 410 [M+H]⁺; HPLC purity 99.1%at 254 nm.

Example 464-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(pyridin-4-ylmethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(WZ-141-89)

The compound WZ141-89 was synthesized by employing the above similarmethod for preparation of4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-methyl-4H-thieno[3,2-b]pyrrole-5-carboxamide(WZ141-84). ¹H NMR (300 MHz, DMSO-d₆) δ 8.79 (t, 1H), 8.46 (d, 2H),7.45-7.05 (m, 8H), 4.92 (s, 2H), 4.41 (d, 2H), 3.58 (m, 2H), 2.32 (s,3H), 1.10 (t, 3H); ESI MS m/z 433 [M+H]+; HPLC purity 94.2% at 254 nm.

Example 474-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(3-morpholinopropyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(WZ-141-90)

The compound WZ141-90 was synthesized by employing the similar methodfor preparation of4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-methyl-4H-thieno[3,2-b]pyrrole-5-carboxamide(WZ141-84). ¹H NMR (300 MHz, DMSO-d₆) δ 8.12 (d, 2H), 7.4-7.0 (m, 6H),4.93 (s, 2H), 3.7-3.4 (m, 6H), 3.4-3.2 (m, 2H), 2.4-2.2 (s, 9H), 1.7-1.5(m, 2H), 1.10 (t, 3H); ESI MS m/z 469 [M+H]⁺; HPLC purity 98.0% at 254nm.

Example 48N-ethyl-2-(5-(4-methylpiperazine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide(WZ-141-91)

The compound WZ141-91 was synthesized by employing the similar methodfor preparation of4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-methyl-4H-thieno[3,2-b]pyrrole-5-carboxamide(WZ141-84). ¹H NMR (300 MHz, DMSO-d₆) δ 7.44 (d, 1H), 7.3-7.1 (m, 3H),7.03 (d, 1H), 6.26 (s, 1H), 4.68 (s, 2H), 3.7-3.5 (m, 6H), 2.35 (s, 3H),2.32 (m, 4H), 2.18 (s, 3H), 1.10 (t, 3H); ESI MS m/z 425 [M+H]⁻; HPLCpurity 96.0% at 254 nm.

Example 49 Syntheses of Compounds of Formula (Ic)

Compounds of formula (Ic) can be generally synthesized according toScheme 4.

Example 50 methyl4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0077)

To a solution of 2-chloro-N-(m-tolyl)acetamide (500 mg, 2.7 mmol) inacetonitrile (20 mL), methyl 4H-thieno[3,2-b]pyrrole-5-carboxylate (411mg, 2.3 mmol), potassium carbonate (376 mg, 2.7 mmol) and 18-crown-6 (30mg, 0.11 mmol) was added. The reaction was heated to reflux for 1.5 h.The mixture was diluted with ethyl acetate and water, and the layerswere separated. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was then concentrated. The residuewas purified by column chromatography (silica, heptane/ethyl acetate) toafford the desired product (530 mg, 71%) as a light brown solid. ¹H NMR(300 MHz, DMSO-d₆) δ 10.21 (s, 1H), 7.57 (d, J=5.4, 1H), 7.42 (s, 1H),7.37-7.30 (m, 1H), 7.27 (d, J=5.4, 1H), 7.23 (s, 1H), 7.16 (t, J=7.8,1H), 6.85 (d, J=7.8, 1H), 5.31 (s, 2H), 3.73 (s, 3H), 2.24 (s, 3H); ESIMS m/z 329 [M+H]⁺; HPLC 99.6% (AUC), T_(R) 6.32 min; UV (MeOH) λ_(max)288 nm, ε 29,177.

Example 51N-cyclohexyl-4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0081)

Step 1.4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0080)

Following general procedure A, methyl4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(130 mg, 0.39 mmol) was reacted with lithium hydroxide (47 mg 2.0 mmol)to afford crude product as a light brown solid.

Step 2.N-cyclohexyl-4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0081)

Following general procedure B,4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (120 mg, 0.38 mmol) was reacted with cyclohexylamine (56 mg, 0.57mmol) to afford the desired product (47 mg, 31%) as a white solid. ¹HNMR (300 MHz, DMSO-d₆) δ 10.13 (s, 1H), 7.95 (d, J=8.1, 1H), 7.43-7.38(m, 2H), 7.33 (d, J=8.1, 1H), 7.23-7.10 (m, 3H), 6.84 (d, J=7.5, 1H),5.30 (s, 2H), 3.75-3.58 (m, 1H), 2.24 (s, 3H), 1.85-1.53 (m, 5H),1.35-1.00 (m, 5H); ESI MS m/z 396 [M+H]⁺; HPLC 94.7% (AUC), T_(R) 7.34min; UV (MeOH) λ_(max) 285 nm, ε 28,066.

Example 52N-cyclohexyl-4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0109)

Step 1. N-(2-(2-methoxyethoxy)ethyl)-3-methylaniline (JRW-0101)

To a solution of m-toluidine (1.0 g, 9.3 mmol) in DMF (10 mL),1-bromo-2-(2-methoxyethoxy)ethane (0.85 g, 4.6 mmol) anddiisopropylamine (1.2 g, 0.93 mmol) was added. The mixture was heated to100° C. for 4 h. The mixture was diluted with ethyl acetate and water,and the layers were separated. The organic layer was dried overanhydrous sodium sulfate and filtered. The filtrate was thenconcentrated. The residue was purified by column chromatography (silica,heptane/ethyl acetate) to afford the desired product (650 mg, 66%) as anoil. ¹H NMR (300 MHz, CDCl₃) δ 7.07 (t, J=7.5, 1H), 6.56 (d, J=7.5, 1H),6.52-6.45 (m, 2H), 3.71 (t, J=5.3, 2H), 3.67-3.62 (m, 2H), 3.60-3.52 (m,2H), 3.31 (t, J=5.3, 2H), 2.28 (s, 3H); ESI MS m/z 210 [M+H]⁺.

Step 2. 2-chloro-N-(2-(2-methoxyethoxy)ethyl)-N-(m-tolyl)acetamide(JRW-0104)

To a solution of N-(2-(2-methoxyethoxy)ethyl)-3-methylaniline (650 mg,3.1 mmol) in ethyl acetate (15 mL), water (5 mL) was added. The biphasicsolution was cooled to 0° C. and potassium hydroxide (522 mg, 9.3 mmol)was added in one motion. 2-Chloroacetyl chloride (526 mg, 4.7 mmol) wasadded dropwise over 10 min. The mixture was stirred for 2.5 h, dilutedwith water, and extracted with ethyl acetate. The organic layer wasdried over anhydrous sodium sulfate and filtered. The filtrate was thenconcentrated to afford crude product (830 mg) as light red oil. ¹H NMR(300 MHz, CDCl₃) δ 7.34-7.25 (m, 1H), 7.18 (d, J=7.7, 1H), 7.15-7.03 (m,2H), 3.89 (t, J=5.8, 2H), 3.83 (s, 2H), 3.65 (t, J=5.8, 2H), 3.62-3.55(m, 2H), 3.54-3.46 (m, 2H), 2.37 (s, 3H); ESI MS m/z 286 [M+H]⁺.

Step 3. methyl4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0105)

To a solution of2-chloro-N-(2-(2-methoxyethoxy)ethyl)-N-(m-tolyl)acetamide (830 mg, 2.9mmol) in acetonitrile (20 mL), methyl4H-thieno[3,2-b]pyrrole-5-carboxylate (438 mg, 2.4 mmol), potassiumcarbonate (400 mg, 2.9 mmol) and 18-crown-6 (32 mg, 0.12 mmol) wasadded. The mixture was heated to reflux for 18 h. The mixture wasdiluted with ethyl acetate and water, and the layers were separated. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was then concentrated. The residue was purified by columnchromatography (silica, heptane/ethyl acetate) to afford crude product(1.3 g) as a thick oil. ESI MS m/z 430 [M+H]⁺.

Step 4.4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0107)

Following general procedure A, methyl4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(2.4 mmol) was reacted with lithium hydroxide (287 mg, 12.0 mmol) toafford crude product (1.0 g, quant.) as a light yellow solid. ESI MS m/z417 [M+H]⁺.

Step 5.N-cyclohexyl-4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0109)

Following general procedure B,4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (120 mg, 0.28 mmol) was reacted with cyclohexylamine (42 mg, 0.43mmol) to afford the desired product (120 mg, 83%) as a white gum. ¹H NMR(300 MHz, DMSO-d₆) δ 7.96-7.85 (m, 1H), 7.48-7.30 (m, 4H), 7.30-7.20 (m,1H), 7.17-7.07 (m, 2H), 5.01 (s, 2H), 3.82-3.62 (m, 3H), 3.56-3.35 (m,6H), 3.25 (s, 3H), 2.38 (s, 3H), 1.90-1.54 (m, 5H), 1.41-1.04 (m, 5H);ESI MS m/z 498 [M+H]⁺; HPLC 96.0% (AUC), T_(R) 7.38 min; UV (EtOH)λ_(max) 289 nm, ε 25,434.

Example 53Methyl-trans-4-(4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0110)

Following general procedure B,4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (120 mg, 0.28 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (68 mg, 0.43 mmol) to afford thedesired product (140 mg, 87%) as a white gum. ¹H NMR (300 MHz, DMSO-d₆)δ 7.94 (d, J=7.8, 1H), 7.46-7.20 (m, 5H), 7.15-7.04 (m, 2H), 5.00 (s,2H), 3.78-3.65 (m, 3H), 3.60 (s, 3H), 3.52-3.35 (m, 6H), 3.23 (s, 3H),2.38 (s, 3H), 2.34-2.20 (m, 1H), 2.02-1.81 (m, 4H), 1.53-1.22 (m, 4H);ESI MS m/z 556 [M+H]⁺; HPLC 98.7% (AUC), T_(R) 6.60 min; UV (MeOH)λ_(max) 289 nm, ε 23,567.

Example 54 methyl4-(2-(hexyhm-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0142)

Step 1. 2-chloro-N-hexyl-N-(m-tolyl)acetamide (JRW-0141)

To a solution of N-hexyl-3-methylaniline (500 mg, 2.6 mmol) in ethylacetate (15 mL), water (5 mL) was added. The biphasic solution wascooled to 0° C., and potassium hydroxide (440 mg, 7.8 mmol) added in onemotion. 2-Chloroacetyl chloride (442 mg, 3.9 mmol) was added dropwiseover 10 min. The mixture was stirred for 1 h, diluted with water, andextracted with ethyl acetate. The organic layer was dried over anhydroussodium sulfate and filtered. The filtrate was then concentrated toafford crude product (730 mg) as light red oil. ¹H NMR (300 MHz, CDCl₃)δ 7.32 (t, J=8.0, 1H), 7.24-7.17 (m, 1H), 7.04-6.97 (m, 2H), 3.80 (s,2H), 3.74-3.61 (m, 2H), 2.38 (s, 3H), 1.59-1.41 (m, 2H), 1.37-1.16 (m,6H), 0.92-0.80 (m, 3H); ESI MS m/z 268 [M+H]⁺.

Step 2. methyl4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0142)

To a solution of 2-chloro-N-hexyl-N-(m-tolyl)acetamide (700 mg, 2.6mmol) in acetonitrile (20 mL), methyl4H-thieno[3,2-b]pyrrole-5-carboxylate (394 mg, 2.2 mmol), potassiumcarbonate (361 mg, 2.6 mmol) and 18-crown-6 (29 mg, 0.11 mmol) wasadded. The mixture was heated to reflux for 18 h. The mixture wasdiluted with ethyl acetate and water, and the layers were separated. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was the concentrated. The residue was purified by columnchromatography (silica, heptane/ethyl acetate) to afford the desiredproduct (780 mg, 86%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 7.53(d, J=5.4, 1H), 7.41 (t, J=7.7, 1H), 7.33-7.11 (m, 5H), 4.93 (s, 2H),3.74 (s, 3H), 3.58 (t, J=6.0 Hz, 2H), 2.37 (s, 3H), 1.48-1.30 (m, 2H),1.28-1.15 (m, 6H), 0.81 (t, J=6.7, 3H); ESI MS m/z 413 [M+H]⁺; HPLC97.1% (AUC), T_(R) 7.18 min; UV (EtOH) λ_(max) 289 nm, ε 26,840.

Example 55N-cyclohexyl-4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0148)

Step 1.4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0149)

Following general procedure A, methyl4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(300 mg, 0.73 mmol) was reacted with lithium hydroxide (87 mg, 3.6 mmol)to afford crude product (300 mg) as a light yellow solid. ESI MS m/z 399[M+H]⁺.

Step 2.N-cyclohexyl-4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0148)

Following general procedure B,4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (50 mg, 0.12 mmol) was reacted with cyclohexylamine (15 mg, 0.15mmol) to afford the desired product (60 mg, 99%) as a white foam. ¹H NMR(300 MHz, DMSO-d₆) δ 7.85 (d, J=8.2, 1H), 7.46-7.16 (m, 5H), 7.12-7.06(m, 2H), 4.96 (s, 2H), 3.75-3.60 (m, 1H), 3.56 (t, J=6.7, 2H), 2.36 (s,3H), 1.84-1.52 (m, 5H), 1.43-1.04 (m, 13H), 0.81 (t, J=6.9, 3H); ESI MSm/z 480 [M+H]⁺; HPLC 98.1% (AUC), T_(R) 8.62 min; UV (EtOH) λ_(max) 288nm, ε 24,544.

Example 56methyl-trans-4-(4-(2-(hexyhm-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0149)

Following general procedure B,4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (50 mg, 0.12 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (24 mg, 0.15 mmol) to afford thedesired product (65 mg, 96%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆)δ 7.91 (d, J=8.0, 1H), 7.44-7.33 (m, 2H), 7.33-7.17 (m, 3H), 7.12-7.06(m, 2H), 4.96 (s, 2H), 3.75-3.47 (m, 6H), 2.36 (s, 3H), 2.31-2.20 (m,1H), 1.99-1.78 (m, 4H), 1.51-1.10 (m, 12H), 0.81 (t, J=6.9, 3H); ESI MSm/z 538 [M+H]⁺; HPLC 99.8% (AUC), T_(R) 8.17 min; UV (EtOH) λ_(max) 289nm, ε 26,509.

Example 57Trans-4-(4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (JRW-0260)

Following general procedure A,methyl-trans-4-(4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(58 mg, 0.11 mmol) was reacted with lithium hydroxide (13 mg, 0.54 mmol)to afford the desired product (55 mg, 97%) as a white foam. ¹H NMR (300MHz, DMSO-d₆) δ 12.03 (s, 1H), 7.88 (d, J=7.8, 1H), 7.46-7.17 (m, 5H),7.12-7.06 (m, 2H), 4.97 (s, 2H), 3.74-3.48 (m, 3H), 2.36 (s, 3H),2.19-2.05 (m, 1H), 2.00-1.77 (m, 4H), 1.48-1.09 (m, 12H), 0.88-0.77 (m,3H).; ESI MS m/z 524 [M+H]⁺; HPLC>99% (AUC), T_(R) 7.42 min; UV (EtOH)λ_(max) 288 nm, ε 24,240.

Example 58 methyl4-(2-(benzyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(WZ-141-86)

Step 1. N-benzyl-2-chloro-N-(m-tolyl)acetamide (WZ-141-85)

To the solution of N-benzyl-3-methylaniline (1.35 g, 6.84 mmol) and TEA(0.761 g, 7.53 mmol) in 50 ml methylene chloride, 2-chloroacetylchloride (0.772 g, 0.772 mmol) at 0° C. was slowly added. The resultantmixture was stirred at 0° C. for 30 minutes and then overnight at RT.The mixture was diluted to 100 ml methylene chloride and washed withwater three times, and organic layer was dried over Na₂SO₄. Afterremoving the solvent, the compound was purified by flash columnchromatography using heptane and ethyl acetate as solvent to give theyellowish product in a yield of 74%. ¹H NMR (300 MHz, CD₂Cl₂) δ 7.6-6.7(m, 9H), 4.89 (s, 2H), 3.71 (s, 2H), 2.31 (s, 3H); ESI MS m/z 274[M+H]⁺.

Step 2. methyl4-(2-(benzyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(WZ-141-86)

The mixture of N-benzyl-2-chloro-N-(m-tolyl)acetamide (1.36 g, 4.97mmol), methyl 4H-thieno[3,2-b]pyrrole-5-carboxylate (0.6 g, 3.31 mmol),18-crown-6 ether (0.262 g, 0.993 mmol) and K₂CO₃ (0.915 g, 6.62 mmol) in50 ml of acetonitrile was heated to reflux overnight. Upon cooling, themajority of solvent was removed under vacuum, and the residue wasdissolved in methylene chloride (100 ml) and washed with water. Theorganic layer was dried over Na₂SO₄. After removing solvent, thecompound was purified by flash column using heptane and ethyl acetate assolvent to give the pale white product in a yield of 95%. ¹H NMR (300MHz, DMSO-d₆) δ 7.57 (s, 1H), 7.4-7.1 (m, 11H), 5.06 (s, 2H), 4.83 (s,2H), 3.79 (s, 3H), 2.31 (s, 3H); ESI MS m/z 419 [M+H]⁺; HPLC purity94.3% at 254 nm.

Example 59 Methyltrans-4-(4-(2-(ethyl(phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0318)

Step 1. methyl4-(2-(tert-butoxy)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0277)

To a solution of methyl 4H-thieno[3,2-b]pyrrole-5-carboxylate (1.4 g,7.73 mmol) in acetonitrile (30 mL), potassium carbonate (1.28 g, 9.28mmol), 18-crown-6 ether (102 mg, 0.38 mmol), and tert-butyl2-bromoacetate (1.81 g, 9.28 mmol)was added. The suspension was heatedto 75° C. for 18 h. The mixture was diluted with ethyl acetate andwater, and the layers were separated. The organic layer was dried overanhydrous sodium sulfate and filtered. The filtrate was thenconcentrated. The residue was purified by column chromatography (silica,heptane/ethyl acetate) to afford the desired product (2.1 g, 91%) as alight yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 7.34 (d, J=5.4, 1H), 7.22(d, J=0.7, 1H), 6.87 (dd, J=0.7, 5.4, 1H), 5.11 (s, 2H), 3.84 (s, 3H),1.46 (s, 9H); ESI MS m/z 296 [M+H]+.

Step 2. 2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid(JRW-0288)

To a solution of methyl4-(2-(tert-butoxy)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(1.92 g, 6.50 mmol) in DCM (20 mL), trifluoroacetic acid (2 mL) wasadded. The solution stirred at RT for 4 h, then the mixture wasconcentrated. Toluene was added to the residue and concentrated toobtained crude product as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 7.38(d, J=5.4, 1H), 7.24 (s, 1H), 6.89 (d, J=5.4, 1H), 5.24 (s, 2H), 3.86(s, 3H); ESI MS m/z 240 [M+H]+.

Step 3. Methyl4-(2-(ethyl(phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0294)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (100 mg,0.42 mmol) in DMF (3 mL), N-ethylaniline (101 mg, 0.84 mmol), HATU (318mg, 0.84 mmol), and diisopropylethylamine (108 mg, 0.84 mmol) was added.The reaction was heated to 75° C. for 18 h. The reaction mixture cooled,diluted with ethyl acetate, and washed with water. The organic layer wasdried over anhydrous sodium sulfate and filtered. The filtrate was thenconcentrated. The residue was purified by column chromatography (silica,heptane/ethyl acetate) to afford the desired product (86 mg, 60%) as awhite solid. ¹H NMR (300 MHz, CDCl₃) δ 7.57-7.21 (m, 6H), 7.16 (s, 1H),6.80 (d, J=5.5, 1H), 4.95 (s, 2H), 3.87-3.68 (m, 5H), 1.13 (t, J=7.2,3H); ESI MS m/z 343 [M+H]⁺.

Step 4.4-(2-(ethyl(phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-309)

Following general procedure A, methyl4-(2-(ethyl(phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(86 mg, 0.25 mmol) was reacted with lithium hydroxide (30 mg, 1.3 mmol)to afford crude product (82 mg) as a light brown solid. ESI MS m/z 329[M+H]⁺.

Step 5. Methyltrans-4-(4-(2-(ethyl(phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0318)

Following general procedure B,4-(2-(ethyl(phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (80 mg, 0.24 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (56 mg, 0.29 mmol) to afford thedesired product (81 mg, 71%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆)δ 7.89 (d, J=7.9, 1H), 7.60-7.28 (m, 6H), 7.04-7.13 (m, 2H), 4.95 (s,2H), 3.73-3.52 (m, 5H), 2.31-2.18 (m, 1H), 2.03-1.78 (m, 4H), 1.50-1.20(m, 4H), 1.07-0.93 (s, 3H); ESI MS m/z 468 [M+H]⁺; HPLC 97.7% (AUC),T_(R) 6.51 min; UV (EtOH) λ_(max) 289 nm, ε 28,274.

Example 60 Methyltrans-4-(4-(2-((3-cyanophenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0321)

Step 1. 2-chloro-N-(3-cyanophenyl)-N-ethylacetamide (JRW-0313)

To a solution of 3-(ethylamino)benzonitrile (60 mg, 0.41 mmol) in ethylacetate (7 mL), water (3 mL) was added. The biphasic solution was cooledto 0° C., and potassium hydroxide (69 mg, 1.2 mmol) was added in onemotion. 2-Chloroacetyl chloride (69 mg, 0.62 mmol) was added dropwiseover 10 min. The mixture was stirred for 2 h, diluted with water, andextracted with ethyl acetate. The organic layer was dried over anhydroussodium sulfate and filtered. The filtrate was then concentrated toafford crude product (98 mg) as an oil. ¹H NMR (300 MHz, CDCl₃) δ7.81-7.42 (m, 4H), 3.84-3.70 (m, 4H), 1.15 (t, J=7.2, 3H); ESI MS m/z223 [M+H]⁺.

Step 2. Methyl4-(2-((3-cyanophenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0316)

To a solution of methyl 4H-thieno[3,2-b]pyrrole-5-carboxylate (68 mg,0.37 mmol) in acetonitrile (5 mL), potassium carbonate (62 mg, 0.45mmol), 18-crown-6 ether (5 mg, 0.019 mmol), and2-chloro-N-(3-cyanophenyl)-N-ethylacetamide (98 mg, 0.45 mmol) wasadded. The suspension was heated to 75° C. for 18 h. The mixture wasdiluted with ethyl acetate and water, and the layers were separated. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was then concentrated. The residue was purified by columnchromatography (silica, heptane/ethyl acetate) to afford the desiredproduct (94 mg, 68%) as a white foam. ¹H NMR (300 MHz, CDCl₃) δ7.71-7.62 (m, 1H), 7.61-7.49 (m, 3H), 7.33 (d, J=5.4, 1H), 7.12 (s, 1H),6.82 (d, J=5.4, 1H), 4.98 (s, 2H), 3.87-3.70 (m, 5H), 1.14 (t, J=7.1,3H); ESI MS m/z 368 [M+H]+.

Step 3.4-(2-((3-cyanophenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0319-1) and4-(2-((3-carbamoylphenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0319-2)

Following general procedure A, methyl4-(2-((3-cyanophenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(90 mg, 0.24 mmol) was reacted with lithium hydroxide (12 mg, 0.49mmol). The two products were separated by column chromatography (silica,dichloromethane/methanol) to afford the nitrile (61 mg) and the amide(31 mg) as white solids. ESI MS m/z 354 [M+H]⁺ and m/z 372 [M+H]⁻.

Step 4. Methyltrans-4-(4-(2-((3-cyanophenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0321)

Following general procedure B,4-(2-((3-cyanophenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (60 mg, 0.17 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (56 mg, 0.29 mmol) to afford thedesired product (68 mg, 81%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆)δ 8.02-7.59 (m, 5H), 7.38 (d, J=5.3, 1H), 7.17-7.03 (m, 2H), 5.02 (s,2H), 3.77-3.55 (m, 6H), 2.32-2.18 (m, 1H), 1.99-1.79 (m, 4H), 1.54-1.20(m, 4H), 1.10-0.95 (m, 3H); ESI MS m/z 493 [M+H]⁺; HPLC 98.3% (AUC),T_(R) 6.12 min; UV (EtOH) λ_(max) 289 nm, ε 26,802.

Example 61 Methyltrans-4-(4-(2-((3-carbamoylphenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0322)

Following general procedure B,4-(2-((3-carbamoylphenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (30 mg, 0.08 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (19 mg, 0.10 mmol) to afford thedesired product (33 mg, 80%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆)δ 8.05 (s, 1H), 7.97-7.83 (m, 3H), 7.72-7.43 (m, 3H), 7.37 (d, J=5.4,1H), 7.12-7.05 (m, 2H), 4.98 (s, 2H), 3.75-3.54 (m, 6H), 2.32-2.18 (m,1H), 1.99-1.79 (m, 4H), 1.53-1.22 (m, 4H), 1.10-0.95 (m, 3H); ESI MS m/z511 [M+H]⁺; HPLC 98.2% (AUC), T_(R) 4.81 min; UV (EtOH) λ_(max) 289 nm,ε 28,223.

Example 62 Methyltrans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0326)

Step 1. Methyl4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0298)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (100 mg,0.42 mmol) in DMF (3 mL), N,3-diethylaniline (94 mg, 0.63 mmol), HATU(318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol) wasadded. The reaction was heated to 85° C. for 18 h. The reaction mixturecooled, diluted with ethyl acetate, and washed with water. The organiclayer was dried over anhydrous sodium sulfate and filtered. The filtratewas then concentrated. The residue was purified by column chromatography(silica, heptane/ethyl acetate) to afford the desired product (107 mg,69%) as an orange foam. ¹H NMR (300 MHz, CDCl₃) δ 7.38 (t, J=7.9, 1H),7.31-7.20 (m, 2H), 7.17-7.10 (m, 3H), 6.81 (d, J=5.4, 1H), 4.98 (s, 2H),3.84-3.67 (m, 5H), 2.71 (q, J=7.6, 2H), 1.29 (t, J=7.6, 3H), 1.13 (t,J=7.2, 3H); ESI MS m/z 371 [M+H]⁺.

Step 2.4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0323)

Following general procedure A, methyl4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(100 mg, 0.27 mmol) was reacted with lithium hydroxide (19 mg, 0.81mmol) to afford crude product (100 mg) as a light yellow solid. ESI MSm/z 357 [M+H]⁺.

Step 3. Methyltrans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0326)

Following general procedure B,4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (100 mg, 0.28 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (65 mg, 0.34 mmol) to afford thedesired product (105 mg, 75%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆)δ 7.90 (d, J=7.9, 1H), 7.51-7.19 (m, 5H), 7.13-7.04 (m, 2H), 4.95 (s,2H), 3.74-3.54 (m, 5H), 2.73-2.61 (m, 2H), 2.32-2.18 (m, 1H), 2.01-1.78(m, 4H), 1.51-1.14 (m, 7H), 1.08-0.95 (s, 3H); ESI MS m/z 496 [M+H⁺;HPLC 97.6% (AUC), T_(R) 7.37 min; UV (MeOH) λ_(max) 288 nm, ε 27,343.

Example 634-(2-(Ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0429)

Step 1.Trans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (JRW-0427)

Following general procedure A, methyltrans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(980 mg, 1.98 mmol) was reacted with lithium hydroxide (142 mg, 5.93mmol) to afford crude product (1.0 g) as a light yellow solid. ESI MSm/z 482 [M+H]⁺.

Step 2.4-(2-(Ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0429)

Following general procedure B,trans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid (200 mg, 0.42 mmol) was reacted with 6-aminohexan-1-ol (73 mg, 0.62mmol) to afford the desired product (200 mg, 83%) as a light yellowfoam. ¹H NMR (300 MHz, DMSO-d₆) δ 7.85 (d, J=8.0, 1H), 7.63 (t, J=5.6,1H), 7.46-7.18 (m, 5H), 7.11-7.04 (m, 2H), 4.96 (s, 2H), 4.27 (t, J=5.2,1H), 3.72-3.55 (m, 3H), 3.40-3.32 (m, 2H), 3.05-2.95 (m, 2H), 2.72-2.62(m, 2H), 2.10-1.97 (m, 1H), 1.89-1.68 (m, 4H), 1.53-1.14 (m, 15H), 1.01(t, J=6.9, 3H); ESI MS m/z 581 [M+H]⁺; HPLC>99% (AUC), T_(R) 5.85 min;UV (MeOH) λ_(max) 288 nm, ε 25,165.

Example 64 Sodium6-(trans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate(JRW-0432)

Step 1.4-(2-(Ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-iodohexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(JRW-0431)

A solution of imidazole (60 mg, 0.88 mmol), triphenylphosphine (230 mg,0.88 mmol), and iodine (223 mg, 0.88 mmol) in THF (10 mL) stirred at RTfor 10 min.4-(2-(Ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(170 mg, 0.29 mmol) dissolved in THF (5 mL) was added. The solution wasstirred for 1 h at RT. The reaction was diluted with ethyl acetate andquenched with a 10% Na₂S₂O₃ solution. The mixture was diluted with ethylacetate and water, and the layers separated. The organic layer waswashed with a 10% Na₂S₂O₃ solution, and the brine dried over anhydroussodium sulfate and filtered. The filtrate was then concentrated. Theresidue was partially purified by column chromatography (silica,dichloromethane/methanol) to afford crude product as a white solid. ESIMS m/z 691 [M+H]⁺.

Step 2. Sodium6-(trans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate(JRW-0432)

To a solution of4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-iodohexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide(0.29 mmol) in ethanol (10 mL), sodium sulfite (184 mg, 1.46 mmol) andwater (10 mL) was added. The mixture was heated to 75° C. for 3.5 h. Thereaction was concentrated, and the residue was purified by columnchromatography (silica, dichloromethane/methanol) to afford the desiredproduct (205 mg, quant) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ7.88 (d, J=8.3, 1H), 7.68 (t, J=5.4, 1H), 7.48-7.19 (m, 5H), 7.12-7.04(m, 2H), 4.95 (s, 2H), 3.71-3.55 (m, 3H), 3.04-2.94 (m, 2H), 2.74-2.61(m, 2H), 2.40-2.32 (m, 2H), 2.11-1.97 (m, 1H), 1.88-1.68 (m, 4H),1.62-1.14 (m, 15H), 1.00 (t, J=6.7, 3H); ESI MS m/z 645 [M+H]⁺; HPLC>99%(AUC), T_(R) 5.07 min; UV (MeOH) λ_(max) 288 nm, ε 18,276.

Example 65 Methyltrans-4-(4-(2-(ethyl(3-methoxyphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0327)

Step 1. Methyl4-(2-(ethyl(3-methoxyphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0299)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (100 mg,0.42 mmol) in DMF (3 mL), N-ethyl-3-methoxyaniline (94 mg, 0.63 mmol),HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol)was added. The reaction was heated to 85° C. for 18 h. The reactionmixture cooled, diluted with ethyl acetate, and washed with water. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was then concentrated. The residue was purified by columnchromatography (silica, heptane/ethyl acetate) to afford the desiredproduct (97 mg, 62%) as an orange foam. ¹H NMR (300 MHz, CDCl₃) δ 7.37(t, J=8.0, 1H), 7.29 (d, J=5.4, 1H), 7.15 (s, 1H), 6.99-6.84 (m, 3H),6.81 (d, J=5.4, 1H), 5.02 (s, 2H), 3.86 (s, 3H), 3.83-3.69 (m, 5H), 1.14(t, J=7.2, 3H); ESI MS m/z 373 [M+H]⁺.

Step 2.4-(2-(ethyl(3-methoxyphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0324)

Following general procedure A, methyl4-(2-(ethyl(3-methoxyphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(95 mg, 0.26 mmol) was reacted with lithium hydroxide (18 mg, 0.77 mmol)to afford crude product (88 mg) as a light yellow solid. ESI MS m/z 359[M+H]⁺.

Step 3. Methyltrans-4-(4-(2-(ethyl(3-methoxyphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0327)

Following general procedure B,4-(2-(ethyl(3-methoxyphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (88 mg, 0.25 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (48 mg, 0.34 mmol) to afford thedesired product (78 mg, 63%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆)δ 7.90 (d, J=8.3, 1H), 7.50-7.31 (m, 2H), 7.16-6.91 (m, 5H), 5.00 (s,2H), 3.81 (s, 3H), 3.75-3.53 (m, 6H), 2.33-2.18 (m, 1H), 2.01-1.77 (m,4H), 1.50-1.22 (m, 4H), 1.08-0.95 (m, 3H); ESI MS m/z 498 [M+H]⁺; HPLC96.4% (AUC), T_(R) 6.69 min; UV (MeOH) λ_(max) 289 nm, ε 28,671

Example 66 Methyltrans-4-(4-(2-(ethyho-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0330)

Step 1. Methyl4-(2-(ethyl(o-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0300)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (100 mg,0.42 mmol) in DMF (3 mL), N-ethyl-2-methylaniline (84 mg, 0.63 mmol),HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol)was added. The reaction was heated to 85° C. for 18 h. The reactionmixture cooled, diluted with ethyl acetate, and washed with water. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was then concentrated. The residue was purified by columnchromatography (silica, heptane/ethyl acetate) to afford the desiredproduct (72 mg, 48%) as a white foam. ¹H NMR (300 MHz, CDCl₃) δ7.43-7.21 (m, 5H), 7.16 (s, 1H), 6.78 (d, J=5.4, 1H), 5.02 (d, J=16.9,1H), 4.74 (d, J=16.9, 1H), 4.15 (dq, J=7.1, 14.2, 1H), 3.81 (s, 3H),3.24 (dq, J=7.1, 14.2, 1H), 1.14 (t, J=7.1, 4H); ESI MS m/z 357 [M+H]⁺.

Step 2.4-(2-(ethyl(o-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0328)

Following general procedure A, methyl4-(2-(ethyl(o-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(70 mg, 0.20 mmol) was reacted with lithium hydroxide (23 mg, 0.98 mmol)to afford crude product (69 mg) as a light yellow solid. ESI MS m/z 343[M+H]⁺.

Step 3. methyltrans-4-(4-(2-(ethyl(o-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0330)

Following general procedure B,4-(2-(ethyl(o-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (69 mg, 0.25 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (46 mg, 0.24 mmol) to afford thedesired product (77 mg, 79%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆)δ 7.91 (d, J=7.9, 1H), 7.49-7.29 (m, 5H), 7.11-6.98 (m, 2H), 4.91 (d,J=16.7, 1H), 4.79 (d, J=16.7, 1H), 4.06-3.90 (m, 1H), 3.72-3.53 (m, 4H),3.14-2.99 (m, 1H), 2.34 (s, 3H), 2.31-2.18 (m, 1H), 2.00-1.78 (m, 4H),1.51-1.14 (m, 4H), 0.99 (t, J=7.1, 3H); ESI MS m/z 482 [M+H]⁺; HPLC92.3% (AUC), T_(R) 6.97 min; UV (MeOH) λ_(max) 288 nm, ε 29,468

Example 67 Methyltrans-4-(4-(2-(6-methyl-3,4-dihydroquinolin-1(211)-yl)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0331)

Step 1. Methyl4-(2-(6-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0301)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (100 mg,0.42 mmol) in DMF (3 mL), 6-methyl-1,2,3,4-tetrahydroquinoline (92 mg,0.63 mmol), HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg,1.25 mmol) was added. The reaction was heated to 85° C. for 18 h. Thereaction mixture cooled, diluted with ethyl acetate, and washed withwater. The organic layer was dried over anhydrous sodium sulfate andfiltered. The filtrate was then concentrated. The residue was purifiedby column chromatography (silica, heptane/ethyl acetate) to afford thedesired product (140 mg, 90%) as a white foam. ¹H NMR (300 MHz, CDCl₃) δ7.31 (d, J=5.4, 1H), 7.18 (s, 1H), 7.08-6.96 (m, 3H), 6.82 (d, J=5.4,1H), 5.39 (s, 2H), 3.86-3.74 (m, 5H), 2.82-2.66 (m, 2H), 2.31 (s, 3H),2.05-1.89 (m, 2H); ESI MS m/z 367 [M+H]⁺.

Step 2.4-(2-(6-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0329)

Following general procedure A, methyl4-(2-(6-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(130 mg, 0.35 mmol) was reacted with lithium hydroxide (42 mg, 1.76mmol) to afford crude product (120 mg) as an orange solid. ESI MS m/z355 [M+H]⁺.

Step 3. Methyltrans-4-(4-(2-(6-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0331)

Following general procedure B,4-(2-(6-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (140 mg, 0.40 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (92 mg, 0.47 mmol) to afford thedesired product (130 mg, 67%) as a white solid. ¹H NMR (300 MHz,DMSO-d₆) δ 7.89 (d, J=7.7, 1H), 7.47 (d, J=7.7, 1H), 7.39 (d, J=5.3,1H), 7.19 (d, J=5.3, 1H), 7.12 (s, 1H), 7.08-6.88 (m, 2H), 5.44 (s, 2H),3.73-3.53 (m, 6H), 2.72 (t, J=6.6, 2H), 2.33-2.16 (m, 4H), 2.00-1.74 (m,6H), 1.50-1.20 (m, 4H); ESI MS m/z 494 [M+H]⁺; HPLC 98.9% (AUC), T_(R)7.16 min; UV (MeOH) λ_(max) 287 nm, ε 26,027.

Example 68 Methyltrans-4-(4-(2-(ethyl(p-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0334)

Step 1. Methyl4-(2-(ethyl(p-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0314)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (100 mg,0.42 mmol) in DMF (3 mL), N-ethyl-4-methylaniline (68 mg, 0.50 mmol),HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol)was added. The reaction was heated to 85° C. for 18 h. The reactionmixture cooled, diluted with ethyl acetate, and washed with water. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was then concentrated. The residue was purified by columnchromatography (silica, heptane/ethyl acetate) to afford the desiredproduct (120 mg, 80%) as a white foam. ¹H NMR (300 MHz, CDCl₃) δ7.33-7.18 (m, 5H), 7.16 (s, 1H), 6.80 (d, J=5.4, 1H), 4.95 (s, 2H), 3.84(s, 3H), 3.74 (q, J=7.2, 3H), 2.40 (s, 3H), 1.12 (t, J=7.2, 3H); ESI MSm/z 357 [M+H]⁺.

Step 2.4-(2-(ethyl(p-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0332)

Following general procedure A, methyl4-(2-(ethyl(p-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(120 mg, 0.34 mmol) was reacted with lithium hydroxide (40 mg, 1.68mmol) to afford crude product (109 mg) as a white solid. ESI MS m/z 343[M+H]⁺.

Step 3. methyltrans-4-(4-(2-(ethyl(p-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0334)

Following general procedure B,4-(2-(ethyl(p-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (109 mg, 0.32 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (74 mg, 0.38 mmol) to afford thedesired product (131 mg, 85%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆)δ 7.88 (d, J=7.9, 1H), 7.41-7.25 (m, 5H), 7.12-7.03 (m, 2H), 4.95 (s,2H), 3.72-3.48 (s, 6H), 2.35 (s, 3H), 2.31-2.19 (s, 1H), 2.01-1.77 (m,4H), 1.52-1.19 (m, 4H), 1.05-0.94 (m, 3H); ESI MS m/z 482 [M+H]⁺; HPLC98.0% (AUC), T_(R) 7.04 min; UV (MeOH) λ_(max) 289 nm, ε 27,490.

Example 69 Methyltrans-4-(4-(2-(ethyl(4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0335)

Step 1. Methyl4-(2-((4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0320)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (150 mg,0.63 mmol) in DMF (3 mL),4-(((tert-butyldimethylsilyl)oxy)methyl)-N-ethylaniline (166 mg, 0.63mmol), HATU (477 mg, 1.25 mmol), and diisopropylethylamine (243 mg, 1.88mmol) was added. The reaction was heated to 85° C. for 30 min. Thereaction mixture cooled, diluted with ethyl acetate, and washed withwater. The organic layer was dried over anhydrous sodium sulfate andfiltered. The filtrate was then concentrated. The residue was purifiedby column chromatography (silica, heptane/ethyl acetate) to afford thedesired product (165 mg, 54%) as a light yellow foam. ¹H NMR (300 MHz,CDCl₃) δ 7.31 (d, J=8.1, 2H), 7.23-7.11 (m, 3H), 7.03 (s, 1H), 6.66 (d,J=5.3, 1H), 4.82 (s, 2H), 4.66 (s, 2H), 3.73-3.55 (m, 5H), 0.99 (t,J=7.2, 3H), 0.83 (s, 9H), 0.00 (s, 6H); ESI MS m/z 487 [M+H]⁺.

Step 2.4-(2-(ethyl(4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0333)

Following general procedure A, methyl4-(2-((4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(165 mg, 0.34 mmol) was reacted with lithium hydroxide (40 mg, 1.68mmol) to afford crude product (110 mg) as a white solid. ESI MS m/z 359[M+H]⁻.

Step 3. Methyltrans-4-(4-(2-(ethyl(4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0335)

Following general procedure B,4-(2-(ethyl(4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (110 mg, 0.31 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (71 mg, 0.37 mmol) to afford thedesired product (169 mg, 91%) as a white solid. ¹H NMR (300 MHz,DMSO-d₆) δ 7.89 (d, J=7.8, 1H), 7.52-7.31 (m, 5H), 7.12-7.05 (m, 2H),5.30-5.21 (m, 1H), 4.95 (s, 2H), 4.54 (d, J=5.6, 2H), 3.74-3.51 (m, 6H),2.32-2.18 (m, 1H), 2.00-1.78 (m, 4H), 1.52-1.21 (m, 4H), 1.05-0.94 (m,3H); ESI MS m/z 498 [M+H]⁺; HPLC 98.7% (AUC), T_(R) 5.13 min; UV (MeOH)λ_(max) 288 nm, ε 24,103.

Example 70 Methyltrans-4-(4-(2-(ethyl(3-isopropylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0460)

Step 1. Methyl4-(2-(ethyl(3-isopropylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0454)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (110 mg,0.46 mmol) in DMF (5 mL), N-ethyl-3-isopropylaniline (112 mg, 0.69mmol), HATU (350 mg, 0.92 mmol), and diisopropylethylamine (178 mg, 1.38mmol) was added. The reaction was heated to 85° C. for 1.5 h. Thereaction mixture cooled, diluted with ethyl acetate, and washed withwater. The organic layer was dried over anhydrous sodium sulfate andfiltered. The filtrate was then concentrated. The residue was purifiedby column chromatography (silica, heptane/ethyl acetate) to afford thedesired product (147 mg, 83%) as a light orange oil. ¹H NMR (300 MHz,CDCl₃) δ 7.45-7.34 (m, 1H), 7.31-7.23 (m, 2H), 7.20-7.11 (m, 3H), 6.80(d, J=5.4, 1H), 4.96 (s, 2H), 3.83-3.71 (m, 5H), 3.05-2.87 (m, 1H),1.35-1.25 (m, 6H), 1.18-1.09 (m, 3H); ESI MS m/z 385 [M+H]⁺.

Step 2.4-(2-(Ethyl(3-isopropylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0457)

Following general procedure A, methyl4-(2-(ethyl(3-isopropylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(145 mg, 0.38 mmol) was reacted with lithium hydroxide (45 mg, 1.89mmol) to afford crude product (134 mg) as a light brown solid. ESI MSm/z 371 [M+H]⁺.

Step 3. Methyltrans-4-(4-(2-(ethyl(3-isopropylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0460)

Following general procedure B,4-(2-(ethyl(3-isopropylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (130 mg, 0.35 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (102 mg, 0.53 mmol) to afford thedesired product (135 mg, 75%) as a white solid. ¹H NMR (300 MHz,DMSO-d₆) δ 7.91 (d, J=7.9, 1H), 7.48-7.33 (m, 3H), 7.32-7.19 (m, 2H),7.12-7.03 (m, 2H), 4.93 (s, 2H), 3.73-3.52 (m, 6H), 3.04-2.87 (m, 1H),2.33-2.18 (m, 1H), 2.00-1.78 (m, 4H), 1.51-1.18 (m, 10H), 1.08-0.95 (m,3H); ESI MS m/z 510 [M+H]⁺; HPLC 98.8% (AUC), T_(R) 7.61 min; UV (MeOH)λ_(max) 289 nm, ε 23,933.

Example 71 Methyltrans-4-(4-(2-(ethyl(3-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0461)

Step 1. Methyl4-(2-((3-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0455)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (120 mg,0.50 mmol) in DMF (5 mL),3-(((tert-butyldimethylsilyl)oxy)methyl)-N-ethylaniline (200 mg, 0.75mmol), HATU (381 mg, 1.0 mmol), and diisopropylethylamine (194 mg, 1.50mmol) was added. The reaction was heated to 85° C. for 1.5 h. Thereaction mixture cooled, diluted with ethyl acetate, and washed withwater. The organic layer was dried over anhydrous sodium sulfate andfiltered. The filtrate was then concentrated. The residue was purifiedby column chromatography (silica, heptane/ethyl acetate) to afford thedesired product (156 mg, 63%) as a colorless oil. ¹H NMR (300 MHz,CDCl₃) δ 7.31 (t, J=7.8, 1H), 7.24-7.17 (m, 1H), 7.18-7.06 (m, 3H), 7.02(s, 1H), 6.66 (d, J=5.2, 1H), 4.82 (s, 2H), 4.66 (s, 2H), 3.71-3.54 (m,5H), 0.99 (t, J=6.9, 3H), 0.83 (s, 9H), 0.00 (s, 6H); ESI MS m/z 487[M+H]⁺.

Step 2.4-(2-(ethyl(3-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0458)

Following general procedure A, methyl4-(2-((3-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(156 mg, 0.32 mmol) was reacted with lithium hydroxide (38 mg, 1.60mmol) to afford crude product (150 mg) as a white solid. ESI MS m/z 359[M+H]⁻.

Step 3. Methyltrans-4-(4-(2-(ethyl(3-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0461)

Following general procedure B,4-(2-(ethyl(3-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (150 mg, 0.42 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (121 mg, 0.63 mmol) to afford thedesired product (126 mg, 60%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆)δ 7.90 (d, J=8.0, 1H), 7.54-7.27 (m, 5H), 7.13-7.03 (m, 2H), 5.33-5.24(s, 1H), 4.96 (s, 2H), 4.56 (d, J=5.5, 2H), 3.73-3.52 (m, 6H), 2.31-2.18(m, 1H), 1.99-1.78 (m, 4H), 1.53-1.21 (m, 4H), 1.07-0.94 (m, 3H); ESI MSm/z 498 [M+H]⁺; HPLC 97.2% (AUC), T_(R) 5.23 min; UV (MeOH) λ_(max) 289nm, ε 25,856.

Example 72 Methyltrans-4-(4-(2-((3-(bromomethyl)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0466)

To a solution of carbon tetrabromide (333 mg, 1.0 mmol) andtriphenylphosphine (263 mg, 1.0 mmol) in THF (5 mL), methyltrans-4-(4-(2-(ethyl(3-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(50 mg, 0.10 mmol) in THF (5 mL) was added. The reaction was stirred atRT for 48 h. The mixture was diluted with DCM and washed with water. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was then concentrated. The residue was purified by columnchromatography (silica, dichloromethane/methanol) to afford the desiredproduct (11 mg, 20%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ7.94-7.86 (m, 1H), 7.72-7.30 (m, 5H), 7.19-6.97 (m, 2H), 4.96 (s, 2H),4.74 (s, 2H), 3.76-3.49 (m, 6H), 2.37-2.17 (m, 1H), 2.05-1.75 (m, 4H),1.55-1.19 (m, 4H), 1.10-0.95 (m, 3H); ESI MS m/z 562 [M+H]⁺; HPLC 87.2%(AUC), T_(R) 6.96 min; UV (MeOH) λ_(max) 289 nm, ε 20,648.

Example 73 Methyltrans-4-(4-(2-((3-(dimethylamino)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0478)

Step 1. Methyl4-(2-((3-(dimethylamino)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0475)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (115 mg,0.48 mmol) in DMF (3 mL), N1-ethyl-N3,N3-dimethylbenzene-1,3-diamine(118 mg, 0.72 mmol), HATU (365 mg, 0.96 mmol), and diisopropylethylamine(186 mg, 1.44 mmol) was added. The reaction was heated to 85° C. for 1h. The reaction mixture cooled, diluted with ethyl acetate, and washedwith water. The organic layer was dried over anhydrous sodium sulfateand filtered. The filtrate was then concentrated. The residue waspurified by column chromatography (silica, heptane/ethyl acetate) toafford the desired product (152 mg, 82%) as a yellow solid. ¹H NMR (300MHz, CDCl₃) δ 7.35-7.23 (m, 2H), 7.14 (s, 1H), 6.85-6.60 (m, 4H), 5.05(s, 3H), 3.89-3.61 (m, 5H), 3.02 (s, 6H), 1.29-0.97 (m, 3H); ESI MS m/z386 [M+H]⁺.

Step 2.4-(2-((3-(Dimethylamino)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0476)

Following general procedure A, methyl4-(2-((3-(dimethylamino)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(160 mg, 0.42 mmol) was reacted with lithium hydroxide (50 mg, 2.1 mmol)to afford crude product (130 mg) as a light green solid. ESI MS m/z 372[M+H]⁺.

Step 3. Methyltrans-4-(4-(2-((3-(dimethylamino)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0478)

Following general procedure B,4-(2-((3-(dimethylamino)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (130 mg, 0.35 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (102 mg, 0.53 mmol) to afford thedesired product (132 mg, 74%) as an off-white solid. ¹H NMR (300 MHz,DMSO-d₆) δ 7.91 (d, J=7.9, 1H), 7.35 (d, J=5.4, 1H), 7.28 (t, J=7.9,1H), 7.11-7.04 (m, 2H), 6.86-6.65 (m, 3H), 5.01 (s, 2H), 3.74-3.51 (m,6H), 2.95 (s, 6H), 2.32-2.19 (s, 1H), 2.01-1.79 (m, 4H), 1.49-1.25 (m,4H), 1.00 (t, J=7.1, 3H); ESI MS m/z 511 [M+H]⁺; HPLC 97.8% (AUC), T_(R)5.21 min; UV (MeOH) λ_(max) 289 nm, ε 29,909.

Example 74 Methyltrans-4-(4-(2-(ethyl(3-isobutylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0508)

Step 1. methyl4-(2-(ethyl(3-isobutylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(JRW-0502)

To a solution of2-(5-(methoxycarbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)acetic acid (100 mg,0.42 mmol) in DMF (3 mL), N-ethyl-3-isobutylaniline (89 mg, 0.50 mmol),HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol)was added. The reaction was heated to 85° C. for 4 h. The reactionmixture cooled, diluted with ethyl acetate, and washed with water. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was then concentrated. The residue was purified by columnchromatography (silica, heptane/ethyl acetate) to afford the desiredproduct (128 mg, 77%) as a thick oil. ¹H NMR (300 MHz, CDCl₃) δ 7.38 (t,J=7.7, 1H), 7.28 (d, J=5.4, 1H), 7.21-7.13 (m, 3H), 7.13-7.09 (m, 1H),6.79 (d, J=4.8, 1H), 4.95 (s, 2H), 3.84-3.69 (m, 5H), 2.54 (d, J=7.2,2H), 1.98-1.82 (m, 1H), 1.13 (t, J=7.2, 3H), 0.92 (t, J=6.1, 6H); ESI MSm/z 399 [M+H]⁺.

Step 2.4-(2-(ethyl(3-isobutylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (JRW-0505)

Following general procedure A, methyl4-(2-(ethyl(3-isobutylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate(120 mg, 0.30 mmol) was reacted with lithium hydroxide (36 mg, 1.5 mmol)to afford crude product (125 mg) as a light yellow solid. ESI MS m/z 385[M+H]⁺.

Step 3. Methyltrans-4-(4-(2-(ethyl(3-isobutylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0508)

Following general procedure B,4-(2-(ethyl(3-isobutylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylicacid (120 mg, 0.42 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (90 mg, 0.47 mmol) to afford thedesired product (130 mg, 80%) as a white solid. ¹H NMR (300 MHz,DMSO-d₆) δ 7.90 (d, J=7.9, 1H), 7.47-7.13 (m, 5H), 7.12-7.04 (m, 2H),4.93 (s, 2H), 3.72-3.52 (m, 6H), 2.57-2.46 (m, 2H), 2.32-2.19 (m, 1H),2.00-1.79 (m, 5H), 1.50-1.20 (m, 4H), 1.06-0.94 (m, 3H), 0.86 (d, J=6.6,6H); ESI MS m/z 524 [M+H]⁺; HPLC>99% (AUC), T_(R) 6.73 min; UV (MeOH)λ_(max) 289 nm, ε 19,115.

Example 75 Methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate(JRW-0355)

Step 1. Methyl1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-indole-2-carboxylate(JRW-0349)

To a solution of methyl 1H-indole-2-carboxylate (100 mg, 0.57 mmol) inacetonitrile (5 mL), potassium carbonate (94 mg, 0.68 mmol), 18-crown-6ether (7 mg, 0.03 mmol), and 2-chloro-N-ethyl-N-(m-tolyl)acetamide (120mg, 0.57 mmol) was added. The suspension was heated to 75° C. for 18 h.The mixture was diluted with ethyl acetate and water, and the layerswere separated. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was then concentrated. The residuewas purified by column chromatography (silica, heptane/ethyl acetate) toafford the desired product (108 mg, 54%) as a white solid. ¹H NMR (300MHz, CDCl₃) δ 7.65 (d, J=8.0, 1H), 7.44-7.07 (m, 8H), 5.05 (s, 2H), 3.88(s, 3H), 3.78-3.68 (m, 2H), 2.43 (s, 3H), 1.12 (t, J=7.2, 3H); ESI MSm/z 351 [M+H]+.

Step 2. 1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-indole-2-carboxylicacid (JRW-0353)

Following general procedure A, methyl1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-indole-2-carboxylate (108 mg,0.31 mmol) was reacted with lithium hydroxide (37 mg, 1.54 mmol) toafford crude product (103 mg) as a white solid. ESI MS m/z 337 [M+H]⁺.

Step 3. Methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate(JRW-0355)

Following general procedure B,1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-indole-2-carboxylic acid (103mg, 0.31 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (71 mg, 0.37 mmol) to afford thedesired product (133 mg, 91%) as a white foam. ESI MS m/z 476 [M+H]⁺.

Example 76 Methyltrans-4-(1-(2-(ethyl(-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate(JRW-0424)

Step 1. 2-chloro-N-ethyl-N-(3-ethylphenyl)acetamide (JRW-0413)

To a solution of N-ethyl-3-ethylaniline (0.97 g, 6.50 mmol) in ethylacetate (30 mL), water (10 mL) was added. The biphasic solution wascooled to 0° C., and potassium hydroxide (1.09 g, 19.5 mmol) added inone motion. 2-Chloroacetyl chloride (1.10 g, 0.76 mL, 9.75 mmol) wasadded dropwise over 10 min. The mixture was stirred for 1 h, dilutedwith water, and extracted with ethyl acetate. The organic layer wasdried over anhydrous sodium sulfate and filtered. The filtrate was thenconcentrated to afford crude product (1.54 g) as a mobile oil. ESI MSm/z 226 [M+H]⁺.

Step 2. methyl1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxylate(JRW-0416)

To a solution of methyl 1H-indole-2-carboxylate (582 mg, 3.3 mmol) inacetonitrile (20 mL), potassium carbonate (551 mg, 4.0 mmol), 18-crown-6ether (44 mg, 0.17 mmol), 2-chloro-N-ethyl-N-(3-ethylphenyl)acetamide(750 mg, 3.3 mmol) was added. The suspension was heated to 75° C. for 18h. The mixture was diluted with ethyl acetate and water, and the layerswere separated. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was then concentrated. The residuewas purified by column chromatography (silica, heptane/ethyl acetate) toafford the desired product (0.74 g, 61%) as a light brown solid. ¹H NMR(300 MHz, CDCl₃) δ 7.65 (d, J=8.1, 1H), 7.50-7.07 (m, 8H), 5.06 (s, 2H),3.87 (s, 3H), 3.82-3.69 (m, 4H), 2.78-2.63 (m, 2H), 1.35-1.24 (m, 3H),1.18-1.06 (m, 3H); ESI MS m/z 365 [M+H]⁺.

Step 3.1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxylic acid(JRW-0419)

Following general procedure A, methyl1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxylate(740 mg, 2.0 mmol) was reacted with lithium hydroxide (243 mg, 10.1mmol) to afford crude product (690 mg) as a white solid. ESI MS m/z 350[M+H]⁺.

Step 4. methyltrans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate(JRW-0424)

Following general procedure B,1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxylic acid(690 mg, 1.97 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (457 mg, 2.36 mmol) to afford thedesired product (760 mg, 79%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆)δ 8.20 (d, J=7.9, 1H), 7.58 (d, J=7.9, 1H), 7.51-7.13 (m, 6H), 7.13-7.02(m, 2H), 5.02 (s, 2H), 3.77-3.54 (m, 6H), 2.69 (d, J=7.6, 2H), 2.33-2.21(m, 1H), 2.02-1.81 (m, 4H), 1.52-1.30 (m, 4H), 1.23 (t, J=7.6, 3H),1.08-0.95 (m, 3H); ESI MS m/z 490 [M+H]⁺; HPLC>99% (AUC), T_(R) 7.56min; UV (MeOH) λ_(max) 291 nm, ε 15,737

Example 771-(2-(Ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-1H-indole-2-carboxamide(JRW-0430)

Step 1.trans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylicacid (JRW-0428)

Following general procedure A, methyltrans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate(720 mg, 1.47 mmol) was reacted with lithium hydroxide (105 mg, 4.4mmol) to afford crude product (680 mg) as a white solid. ESI MS m/z 476[M+H]⁺.

Step 2.1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-1H-indole-2-carboxamide(JRW-0430)

Following general procedure B,trans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylicacid (200 mg, 0.42 mmol) was reacted with 6-aminohexan-1-ol (74 mg, 0.63mmol) to afford the desired product (218 mg, 79%) as a light yellowfoam. ¹H NMR (300 MHz, DMSO-d₆) δ 8.17 (d, J=8.0, 1H), 7.69-7.54 (m,2H), 7.50-7.14 (m, 6H), 7.13-7.02 (m, 2H), 5.02 (s, 2H), 4.27 (t, J=5.2,1H), 3.77-3.52 (s, 3H), 3.36 (dd, J=6.4, 11.7, 2H), 3.05-2.95 (m, 2H),2.74-2.63 (m, 2H), 2.13-1.97 (m, 1H), 1.91-1.69 (m, 4H), 1.54-1.15 (m,15H), 1.09-0.93 (m, 3H) ; ESI MS m/z 575 [M+H]⁺; HPLC>99% (AUC), T_(R)6.12 min; UV (MeOH) λ_(max) 291 nm, ε 17,243.

Example 78 Sodium6-(trans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate(JRW-0434)

Step 1.1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-iodohexyl)carbamoyl)cyclohexyl)-1H-indole-2-carboxamide(JRW-0433)

A solution of imidazole (66 mg, 0.97 mmol), triphenylphosphine (254 mg,0.97 mmol), and iodine (246 mg, 0.97 mmol) in THF (10 mL) stirred at RTfor 10 min.1-(2-(Ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-1H-indole-2-carboxamide(186 mg, 0.32 mmol) dissolved in THF (5 mL) was added. The solutionstirred for 1 h at RT. The reaction was diluted with ethyl acetate andquenched with a 10% Na₂S₂O₃ solution. The mixture was diluted with ethylacetate and water, and the layers were separated. The organic layer waswashed with a 10% Na₂S₂O₃ solution, and the brine dried over anhydroussodium sulfate and filtered. The filtrate was then concentrated. Theresidue was partially purified by column chromatography (silica,dichloromethane/methanol) to afford crude product as a white solid. ESIMS m/z 685 [M+H]⁺.

Step 2. sodium6-(trans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate(JRW-0434)

To a solution of1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-iodohexyl)carbamoyl)cyclohexyl)-1H-indole-2-carboxamide(0.32 mmol) in ethanol (10 mL), sodium sulfite (203 mg, 1.6 mmol) andwater (10 mL) was added. The mixture was heated to 75° C. for 2 h. Thereaction was concentrated, and the residue was purified by columnchromatography (silica, dichloromethane/methanol) to afford the desiredproduct (190 mg, 89%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.21(d, J=8.2, 1H), 7.69 (t, J=5.6, 1H), 7.58 (d, J=7.8, 1H), 7.51-7.14 (m,6H), 7.12-7.03 (m, 2H), 5.02 (s, 2H), 3.76-3.53 (m, 3H), 3.00 (dd,J=6.6, 12.7, 2H), 2.76-2.61 (m, 2H), 2.39-2.32 (m, 2H), 2.12-1.99 (m,1H), 1.90-1.70 (m, 4H), 1.61-1.14 (m, 15H), 1.08-0.95 (s, 3H); ESI MSm/z 639 [M+H]⁺; HPLC>99% (AUC), T_(R) 5.07 min; UV (MeOH) λ_(max) 291nm, ε 15,800.

Example 79 Methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6-methoxy-1H-indole-2-carboxamido)cyclohexane-1-carboxylate(JRW-0359)

Step 1. Methyl1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6-methoxy-1H-indole-2-carboxylate(JRW-0351)

To a solution of methyl 6-methoxy-1H-indole-2-carboxylate (116 mg, 0.57mmol) in acetonitrile (5 mL), potassium carbonate (94 mg, 0.68 mmol),18-crown-6 ether (7 mg, 0.03 mmol), and2-chloro-N-ethyl-N-(m-tolyl)acetamide (120 mg, 0.57 mmol) was added. Thesuspension was heated to 70° C. for 2 d. The mixture was diluted withethyl acetate and water, and the layers were separated. The organiclayer was dried over anhydrous sodium sulfate and filtered. The filtratewas then concentrated. The residue was purified by column chromatography(silica, heptane/ethyl acetate) to afford the desired product (155 mg,72%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 7.51 (d, J=8.7, 1H),7.41-7.31 (m, 1H), 7.24-7.08 (m, 4H), 6.79 (dd, J=2.2, 8.7, 1H),6.62-6.55 (m, 1H), 5.01 (s, 2H), 3.87 (s, 3H), 3.84 (s, 3H), 3.81-3.68(m, 2H), 2.42 (s, 3H), 1.18-1.09 (m, 3H); ESI MS m/z 381 [M+H]+.

Step 2.1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6-methoxy-1H-indole-2-carboxylicacid (JRW-0357)

Following general procedure A, methyl1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6-methoxy-1H-indole-2-carboxylate(150 mg, 0.39 mmol) was reacted with lithium hydroxide (47 mg, 1.97mmol) to afford crude product (150 mg) as a white solid. ESI MS m/z 367[M+H]⁺.

Step 3. Methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6-methoxy-1H-indole-2-carboxamido)cyclohexane-1-carboxylate(JRW-0359)

Following general procedure B,1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6-methoxy-1H-indole-2-carboxylicacid (150 mg, 0.41 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (95 mg, 0.49 mmol) to afford thedesired product (163 mg, 78%) as a white foam. ESI MS m/z 506 [M+H]⁺.

Example 80 Methyltrans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-furo[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-360)

Step 1. Methyl4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-furo[3,2-b]pyrrole-5-carboxylate(JRW-0352)

To a solution of methyl 4H-furo[3,2-b]pyrrole-5-carboxylate (94 mg, 0.57mmol) in acetonitrile (5 mL), potassium carbonate (94 mg, 0.68 mmol),18-crown-6 ether (7 mg, 0.03 mmol), and2-chloro-N-ethyl-N-(m-tolyl)acetamide (120 mg, 0.57 mmol) was added. Thesuspension was heated to 70° C. for 2 d. The mixture was diluted withethyl acetate and water, and the layers were separated. The organiclayer was dried over anhydrous sodium sulfate and filtered. The filtratewas then concentrated. The residue was purified by column chromatography(silica, heptane/ethyl acetate) to afford the desired product (166 mg,86%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 7.47 (d, J=2.2, 1H),7.35 (t, J=7.7, 1H), 7.24-7.17 (m, 1H), 7.16-7.07 (m, 2H), 6.81-6.78 (m,1H), 6.38-6.36 (m, 1H), 4.86 (s, 2H), 3.85-3.67 (m, 5H), 2.40 (s, 3H),1.12 (t, J=7.2, 3H); ESI MS m/z 341 [M+H]+.

Step 2.4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-furo[3,2-b]pyrrole-5-carboxylicacid (JRW-358)

Following general procedure A, methyl4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-furo[3,2-b]pyrrole-5-carboxylate(160 mg, 0.47 mmol) was reacted with lithium hydroxide (56 mg, 2.35mmol) to afford crude product (150 mg) as a white solid. ESI MS m/z 327[M+H]⁺.

Step 3. Methyltrans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-furo[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-360)

Following general procedure B,4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-furo[3,2-b]pyrrole-5-carboxylicacid (150 mg, 0.46 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (106 mg, 0.55 mmol) to afford thedesired product (178 mg, 83%) as a white foam. ESI MS m/z 466 [M+H]⁺.

Example 81 Methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-pyrrole-2-carboxamido)cyclohexane-1-carboxylate(JRW-0456)

Step 1. methyl1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-pyrrole-2-carboxylate(JRW-0450)

To a solution of methyl 1H-pyrrole-2-carboxylate (295 mg, 2.36 mmol) inacetonitrile (20 mL), potassium carbonate (391 mg, 2.83 mmol),18-crown-6 ether (31 mg, 0.12 mmol), and2-chloro-N-ethyl-N-(m-tolyl)acetamide (500 mg, 2.36 mmol) was added. Thesuspension was heated to 75° C. for 18 h. The mixture was diluted withethyl acetate and water, and the layers were separated. The organiclayer was dried over anhydrous sodium sulfate and filtered. The filtratewas then concentrated. The residue was purified by column chromatography(silica, heptane/ethyl acetate) to afford the desired product (530 mg,75%) as a clear oil. ¹H NMR (300 MHz, CDCl₃) δ 7.35 (t, J=7.5, 1H),7.24-7.10 (m, 3H), 6.93 (dd, J=1.8, 3.9, 1H), 6.78-6.70 (m, 1H), 6.13(dd, J=2.6, 3.9, 1H), 4.77 (s, 2H), 3.85-3.61 (m, 6H), 2.41 (s, 3H),1.13 (t, J=7.2, 3H); ESI MS m/z 301 [M+H]+.

Step 2. 1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-pyrrole-2-carboxylicacid (JRW-0453)

Following general procedure A, methyl1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-pyrrole-2-carboxylate (530 mg,1.76 mmol) was reacted with lithium hydroxide (211 mg, 8.82 mmol) toafford crude product (475 mg) as a white solid. ESI MS m/z 287 [M+H]⁺.

Step 3. methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-pyrrole-2-carboxamido)cyclohexane-1-carboxylate(JRW-0456)

Following general procedure B,1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-pyrrole-2-carboxylic acid (475mg, 1.66 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (482 mg, 2.49 mmol) to afford thedesired product (505 mg, 71%) as a white foam. ¹H NMR (300 MHz, DMSO-d₆)δ 7.62 (d, J=8.0, 1H), 7.41-7.30 (m, 1H), 7.26-7.12 (m, 3H), 6.82-6.67(m, 2H), 5.94 (dd, J=2.6, 3.8, 1H), 4.77 (s, 2H), 3.67-3.54 (m, 6H),2.34 (s, 3H), 2.29-2.18 (s, 1H), 1.98-1.74 (m, 4H), 1.48-1.26 (m, 4H),1.09-0.92 (m, 3H); ESI MS m/z 426 [M+H]⁺; HPLC 97.5% (AUC), T_(R) 6.05min; UV (MeOH) λ_(max) 264 nm, ε 11,978.

Example 82 Methyltrans-4-(6-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-611-thieno[2,3-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0463)

Step 1. Methyl6-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6H-thieno[2,3-b]pyrrole-5-carboxylate(JRW-0459)

To a solution of methyl 6H-thieno[2,3-b]pyrrole-5-carboxylate (171 mg,0.94 mmol) in acetonitrile (10 mL), potassium carbonate (157 mg, 1.1mmol), 18-crown-6 ether (13 mg, 0.047 mmol), and2-chloro-N-ethyl-N-(m-tolyl)acetamide (200 mg, 0.94 mmol) was added. Thesuspension was heated to 75° C. for 18 h. The mixture was diluted withethyl acetate and water, and the layers were separated. The organiclayer was dried over anhydrous sodium sulfate and filtered. The filtratewas then concentrated. The residue was purified by column chromatography(silica, heptane/ethyl acetate) to afford the desired product (260 mg,77%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 7.37 (t, J=7.6, 1H),7.25-7.09 (m, 4H), 6.99-6.95 (m, 1H), 6.91-6.85 (m, 1H), 4.93 (s, 2H),3.92-3.64 (m, 5H), 2.43 (s, 3H), 1.14 (t, J=7.2, 4H); ESI MS m/z 357[M+H]+.

Step 2.6-(2-(Ethyl(m-tolyl)amino)-2-oxoethyl)-6H-thieno[2,3-b]pyrrole-5-carboxylicacid (JRW-0462)

Following general procedure A, methyl6-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6H-thieno[2,3-b]pyrrole-5-carboxylate(260 mg, 0.73 mmol) was reacted with lithium hydroxide (87 mg, 3.6 mmol)to afford crude product (240 mg) as a white solid. ESI MS m/z 342[M+H]⁺.

Step 3. Methyltrans-4-(6-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6H-thieno[2,3-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate(JRW-0463)

Following general procedure B,6-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6H-thieno[2,3-b]pyrrole-5-carboxylicacid (240 mg, 0.70 mmol) was reacted with methyltrans-4-aminocyclohexane-1-carboxylate (203 mg, 1.05 mmol) to afford thedesired product (320 mg, 95%) as a light yellow foam. ¹H NMR (300 MHz,DMSO-d₆) δ 7.88 (d, J=8.0, 1H), 7.44-7.33 (m, 1H), 7.28-7.14 (m, 3H),7.11-6.95 (m, 3H), 4.90 (s, 2H), 3.72-3.52 (m, 6H), 2.36 (s, 3H), 2.25(s, 1H), 2.02-1.76 (m, 4H), 1.51-1.20 (m, 4H), 1.00 (t, J=6.9, 3H); ESIMS m/z 482 [M+H]⁺; HPLC>99% (AUC), T_(R) 7.01 min; UV (MeOH) λ_(max) 287nm, ε 12,894.

Example 83 Inhibition of Nluc Luciferase

K562 were plated into wells of a Corning 3707 assay plate in 20 μL RPMImedia at 2000 cells/well and incubated overnight. A Trail titration wasprepared in 10 μL RPMI media and added to cells. Then either 10 μL 4×REALTIME-GLO™ MT Cell Viability reagent (RTCV; Promega Corporation) ormedia (control) was added to each well. The reactions were incubated at37° C./5% CO₂ at various time points, and luminescence was measured on aTecan M1000 Pro plate reader. At 5 hrs, 40 μL CASPASE-GLO® 3/7 detectionreagent (Promega Corporation) was added with or without 200 μM JRW-0004.The reaction was incubated for 2 hrs at room temperature (RT), andluminescence was measured on the Tecan M1000 Pro plate reader. FIG. 1demonstrates that JRW-0004 can inhibit the Nluc enzyme in REALTIME-GLO™MT Cell Viability reagent, and therefore recover the CASPASE-GLO® assaywindow in a multiplex that combines REALTIME-GLO™ assay and CASPASE-GLO®assay.

Example 84 Inhibition of Purified Nluc Luciferase

In a Corning 3570 assay plate, purified Nluc enzyme (PromegaCorporation), 20 mM DTT, and an Nluc pro-substrate (PBI-4442, which isdescribed in US Patent Publication No. 2013/0130289), which is convertedto an Nluc substrate upon reduction with DTT, was added. Compoundtitrations of JRW-0004, JRW-0013, JRW-0006, JRW-0042, JRW-0138, andJRW-0147 were then made (1:2 serial dilution in PBS, 11 points plus noinhibitor control, starting at 200 μM; 100 μM final concentration inreaction). Compound titrations were added in equal volume to the wellsof the assay plate. The reactions were incubated at RT for 2 hrs, andluminescence was measured on Tecan M1000 Pro plate reader. Half-maximalinhibitor (thienopyrrole compound) concentrations (IC₅₀s) weredetermined using GraphPad Prism 6.03. FIG. 2 demonstrates that allcompounds are capable of inhibiting purified Nluc enzyme.

Example 85 Specificity of Inhibition of Nluc Luciferase

The following example describes the specificity of disclosedthienopyrrole compounds, JRW-0004, JRW-0013, JRW-0006, JRW-0042,JRW-0138, and JRW-0147, to inhibit Nluc luciferase activity versusfirefly luciferase activity, e.g., ULTRAGLO® luciferase. In a Corning3570 assay plate, a solution containing 1 μM luciferin in LuciferaseDetection Reagent (Promega Corporation V865/859) was added to the assaywells. An equal volume of titrations the thienopyrrole compounds,JRW-0004, JRW-0013, JRW-0006, JRW-0042, JRW-0138, and JRW-0147 were thenadded to the wells. The reactions were incubated at RT for 2 hrs, andluminescence was measured on Tecan M1000 Pro plate reader. FIG. 3demonstrates that the thienopyrrole compounds did not inhibit thefirefly luciferase activity.

Example 86 Thienopyrrole Compounds Enable Multiplexing

The following examples describe the use of the thienopyrrole compoundsof the present invention to allow multiplexing of assays which utilizingNluc luciferase and another luciferase, e.g., firefly luciferase.

A) In a Corning 3570 assay plate, MCF7 cells were plated at 1,000cells/well in 20 μL of cell culture media (EMEM supplemented with 0.01mg/mL human recombinant insulin and 10% fetal bovine serum) andincubated in overnight. 10 μL of 4× REALTIME-GLO™ MT Cell Viabilityreagents (Promega Corporation) in media or 10 μL media only was added tothe cells. 10 μL of 40 μM staurosporine in media or 10 μL media only(control) was added to the cells. The cells were incubated, and caspaseactivation monitored at 5.5 hrs post-reagent addition. To monitorcaspase activation, 40 μL CASPASE-GLO® 3/7 Assay Reagent (PromegaCorporation), either on its own (Caspase) or with 200 μM of JRW-0004,JRW-0013, or JRW-0042, was added. Reactions were incubated at RT, andluminescence was measured at 1 hr 10 min post-reagent addition on TecanM1000 Pro plate reader.

Table 1 shows that the compounds inhibit the background luminescencefrom REALTIME-GLO™ in the multiplex with CASPASE-GLO® (media containingREALTIME-GLO™). When comparing the signal generated from theCASPASE-GLO® reagent in media only versus the signal generated in mediacontaining REALTIME-GLO™ reagent, the signal is higher in the multiplexreaction. The compounds inhibit the Nluc enzyme and decrease thebackground luminescence from REALTIME-GLO™ reagent.

TABLE 1 MCF7 Media containing Media only RealTime-Glo Control 10 μMStauro Control 10 μM Stauro Avg SD Avg SD Avg SD Avg SD Caspase 74481076 51670 7299 38144 3853 85801 6888 Fold Cont 6.94 2.25 JRW-0004 92611017 54589 9231 9090 161 50266 3766 Fold Cont 5.89 5.53 JRW-0013 80692461 55282 13491 8636 1777 54338 6157 Fold Cont 6.85 6.29 JRW-0042 107181269 62280 6594 10679 1747 53956 4432 Fold Cont 5.81 5.05

B) A549 cells were plated at 1,000 cells/well in 20 μL F12K media intowells of a Corning 3570 plate (n-4) and incubated overnight. Then,REALTIME-GLO™ MT Cell Viability assay reagents (Promega Corporation)were added to the wells as a 2× solution in 20 μL media. The reactionwas incubated for 1 hr. 40 μL of a titration of JRW-0013 or JRW-0147 inCASPASE-GLO® 3/7 assay reagent (Promega Corporation) at 2×concentrations was added. The reactions were incubated at RT, andluminescence was determined at 1 hr.

Table 2 demonstrates that these compounds can inhibit the Nluc enzyme inREALTIME-GLO™ MT Cell Viability assay in a multiplex with a CASPASE-GLO®assay in a dose-dependent manner.

TABLE 2 Inhibitor Caspase, JRW-0013 Caspase, JRW-0147 μM, final Avg SD %remaining Avg SD % remaining 100 3619 454  6.10% 3389 177  5.71% 66.674017 450  6.77% 3427 51  5.78% 44.44 4419 35  7.45% 4367 189  7.36%29.63 5132 145  8.65% 4394 331  7.41% 19.75 5095 330  8.59% 5302 223 8.94% 13.17 6014 591  10.14% 5545 401  9.35% 8.78 6703 406  11.30% 7125533  12.01% 5.85 8059 801  13.59% 8013 375  13.51% 3.90 9148 271  15.42%9790 1213  16.51% 2.60 11813 679  19.92% 11677 189  19.69% 1.73 128791093  21.71% 15580 1288  26.27% 0 59313 1970 100.00% 59313 1970 100.00%

Example 87

The following example demonstrates that compounds JRW-0004, WZ141-88,WZ141-86, WZ141-74, WZ141-84, WZ141-89, WZ141-90, and WZ141-91 inhibitNluc luciferase. A solution of purified Nluc and an Nluc pro-substrate(PBI-4442, which is described in US 2013/0130289), which is converted toan Nluc substrate upon reduction with DTT, were prepared in PBS buffer,pH 7.5. A 40 mM solution of DTT in PBS, pH 7.5 containing 1% TERGITOLwas also prepared. Titrations of the thienopyrrole compounds JRW-0004,WZ141-88, WZ141-86, WZ141-74, WZ141-84, WZ141-89, WZ141-90, and WZ141-91in the buffer containing DTT and TERGITOL were then prepared. An equalvolume of the Nluc/pro-substrate solution was added to the thienopyrrolecompound titrations in wells of an assay plate. The reactions wereincubated at RT, and luminescence was measured at various time points onthe Tecan M100 Pro plate reader (integration time 200 ms). IC₅₀ valueswere determined using GraphPad Prism 6.03. FIGS. 4-6 demonstrate thatall compounds tested inhibit Nluc luciferase in a dose- andtime-dependent manner.

Example 88 Cell Permeability

The following example demonstrates the permeability of the thienopyrrolecompounds described herein. HEK293 or HeLa cells were transientlytransfected with Beta-2 Adrenergic Receptor-Nluc (B2AR-Nluc) fusionprotein to anchor and orient Nluc cytosolically. 24 hrspost-transfection, cells were treated with −/+50 μg/mL digitonin toemulate a live and lytic scenario, respectively. Then, intact cells andpermeabilized cells were exposed to a compound response curve ofthienopyrrole compound up to 2 hrs. 10 μM furimazine (Promega Corp.) wasadded, and luminescence was measured. If a thienopyrrole compound(inhibitor) was permeable, the dose-response curves for both live andpermeabilized cells overlapped. However, if a thienopyrrole compound wasimpermeable, the EC₅₀ was right-shifted in the live cells relative tothe permeabilized cells. See FIGS. 7-11.

Example 89 Inhibition of Extracellular BRET

The following example demonstrates the ability of the thienopyrrolecompounds described herein to inhibit extracellular BRET. See FIG. 12A.HEK293 were transiently transfected with an Nluc-HDAC6 fusion protein toorient Nluc-HDAC6 inside the cell. 24 hrs post-transfection, cells weretreated 10 ng/mL purified Nluc-HALOTAG labeled with NANOBRET 618 ligandto simulate spurious extracellular BRET. Cells were then exposed to acompound response curve of the thienopyrrole compounds, JRW-0013,JRW-0051, JRW-0147, and JRW-0187, for 2 hrs. 10 μM furimazine was thenadded, and BRET ratio 610/450 nm was measured. If a thienopyrrolecompound, such as JRW-0013, was permeable, the BRET ratio remainedconstant across the compound dose-response curve. If a thienopyrrolecompound, such as JRW-0051, JRW-0147, or JRW-0187, was impermeable, theBRET ratio decreased across the compound dose-response curve andenhanced intracellular Nluc signal while inhibiting extracellular BRET.See FIGS. 12B-12E.

Example 90 Inhibition of Extracellular Luciferase Activity andEnhancement of Intracellular BRET

The following example demonstrates the ability of the thienopyrrolecompounds described herein to inhibit extracellular luciferase activitywhile enhancing intracellular BRET. See FIG. 13A. HEK293 weretransiently transfected with Nluc-HALOTAG fusion protein to orientNluc-HALOTAG inside the cell. 24 hrs post-transfection, cells werelabeled with NANOBRET 618 ligand to simulate specific intracellularBRET. Cells were then treated 10 ng/mL purified Nluc to simulatespurious extracellular luminescence. Cells were exposed to a compoundresponse curve of the thienopyrrole compounds, JRW-0013, JRW-0051,JRW-0147, and JRW-0187, for 2 hrs. 10 μM furimazine was added, and theBRET ratio 610/450 nm was measured. If an inhibitor, such as JRW-0013,was permeable, the BRET ratio remained constant across the compounddose-response curve. If an inhibitor, such as JRW-0051, JRW-0147, orJRW-0187, was impermeable, the intracellular BRET ratio was enhancedacross the compound dose-response curve while inhibiting spuriousextracellular Nluc signal. See FIGS. 13B-13F.

Example 91 Cell Permeability with Bioluminescent Imaging

HeLa cells were transiently transfected with Beta-2 AdrenergicReceptor-Nluc (B2AR-Nluc; C-Terminus Nluc) or Nluc-Beta-2 AdrenergicReceptor (Nluc-B2AR; N-terminus Nluc) fusion protein to anchor andorient Nluc intracellularly or extracellularly, respectively. 24 hrspost-transfection, cells were treated with −/+30 μM of a thienopyrrolecompound, JRW-0147, JRW-0051, and JRW-0138. 10 μM furimazine was added,and luminescence was detected by imaging on the Olympus LV200.Thienopyrrole compounds JRW-0147 and JRW-0051 were impermeable andinhibited extracellular Nluc and enhanced intracellular Nluc. CompoundJRW-0138 was cell permeable and inhibited both intracellular andextracellular Nluc. See FIG. 14

Example 92 Characterization of an Impermeable Thienopyrrole Compound ina Target Engagement Model

HEK293 cells were transiently transfected with Src-Nluc fusion protein.See FIG. 15A. 24 hours post-transfection, cells were treated with −/+50μg/mL digitonin to simulate a live and lytic scenario, respectively.Intact cells and permeabilized cells were labeled with 1 μMDasatinib-DY605 tracer (impermeable) and treated with a cell impermeablethienopyrrole compound JRW-0147 response curve for 2 hrs. 10 μMfurimazine was added, and a BRET ratio 610/450 nm was recorded. FIGS.15B-15C demonstrates that JRW-0147 inhibited BRET in cell debris, butnot BRET inside cells.

Example 93 Cell Permeability Time Course

The following example demonstrates the permeability of the thienopyrrolecompounds described herein. HEK293 cells were transiently transfectedwith Nluc luciferase to express it cytosolically. Twenty-four hourspost-transfection, cells were exposed to a compound response curve ofthe thienopyrrole compound JRW-0147 or JRW-0013 for 10 minutes, 30minutes, or 120 minutes. 10 μM furimazine was then added, andluminescence was measured.

If the thienopyrrole compound (inhibitor) was permeable, the compoundwould passively enter the cell and decrease the RLUs from Nluc in adose-dependent manner independent of time, such as was seen by JRW-0013(FIG. 16). If the thienopyrrole compound (inhibitor) was impermeable,the compound would not actively or passively enter the cell, and nosignificant change in RLUs from Nluc would be observed in adose-dependent manner independent of time, such as was seen by JRW-0147(FIG. 16).

Example 94 Measurement of Endocytosis by Chemical Conjugation orMolecular Fusion to Nluc

Antibodies, proteins, receptors, drugs, drug carriers, peptides, sugars,fatty acids, nanoparticles, or other biomolecules could be eitherchemically conjugated or fused to Nluc to measure endocytosis incombination with the cell impermeable thienopyrrole compound (inhibitor)described herein.

For example, a monoclonal antibody (e.g., Nluc-Trastuzumab) could bechemically conjugated and bound to the HER2 receptor expressed on thesurface of SKBR3 cells. Cell impermeable Nluc inhibitor could be appliedto inhibit extracellular Nluc-Trastuzumab. Upon addition of acoelenterazine substrate, a gain of signal assay could be used tokinetically measure active/passively internalized Trastuzumab-Nluc-HER2receptor, which can be extended to other antibodies, proteins,receptors, drugs, drug carriers, peptides, sugars, fatty acids,nanoparticles, or other biomolecules chemically conjugated to Nluc.

In another example, Nluc-GPCRs (e.g., Nluc-B2AR) could be geneticallyfused and expressed in mammalian cells. A cell impermeable thienopyrrolecompound (inhibitor) as described herein could be applied to inhibitextracellular or membrane bound Nluc-B2AR. Upon addition ofcoelenterazine substrate, a gain of signal assay could be used tokinetically measure active/passively internalized or recycled Nluc-GPCR,which can be extended to other proteins or receptors genetically fusedto Nluc.

Example 95 Specificity of Inhibition of Nluc Luciferase

The following example describes the specificity of disclosedthienopyrrole compounds, JRW-0251, JRW-0344, and JRW-0147, to inhibitNluc luciferase activity versus firefly luciferase activity, e.g.,ULTRAGLO® luciferase. In a Corning 3570 assay plate, a solutioncontaining 1 μM luciferin in Luciferase Detection Reagent (PromegaCorporation V865/859) was added to the assay wells. An equal volume oftitrations the thienopyrrole compounds, JRW-0251, JRW-0344, and JRW-0147were then added to the wells. The reactions were incubated at RT for 2hrs, and luminescence was measured on Tecan M1000 Pro plate reader.Table 3 demonstrates that the thienopyrrole compounds did not inhibitthe firefly luciferase activity.

TABLE 3 Nluc [cmpd], JRW-0147 JRW-0251 JRW-0344 μM Avg SD S/B Avg SD S/BAvg SD S/B 200 23722 4826 0.20 23311 208 0.20 16291 559 0.15 100 327027220 0.27 34389 977 0.30 23445 1930 0.21 50 42630 9430 0.36 44219 8090.39 32113 3215 0.29 25 54498 13398 0.46 55210 1757 0.48 40392 3250 0.3612.5 66480 17301 0.56 68664 2710 0.60 52627 5302 0.47 6.25 79191 190700.67 81193 1762 0.71 65136 4742 0.59 3.125 96256 5190 0.81 86555 16250.76 71976 4730 0.65 1.5625 103399 1432 0.87 96602 2280 0.84 83141 60070.75 0.78125 111603 2902 0.94 105881 4664 0.92 95060 6457 0.86 0.390625113278 2111 0.95 106075 788 0.93 99851 5327 0.90 0.1953125 115442 17230.97 108634 436 0.95 102409 6375 0.92 0 119061 5586 1.00 114494 12031.00 110876 6347 1.00 UltraGlo luciferase [cmpd], JRW-0147 JRW-0251JRW-0344 μM Avg SD S/B Avg SD S/B Avg SD S/B 200 78035 4010 0.90 77788618 0.92 77230.33 1326 0.93 100 80636 3824 0.93 79556 842 0.94 79847.67842 0.96 50 81106 3963 0.94 81930 632 0.97 81805.00 791 0.98 25 840333435 0.97 83382 780 0.99 83084.00 711 1.00 12.5 83291 2687 0.97 83569583 0.99 82454.67 723 0.99 6.25 83282 2956 0.97 83665 969 0.99 83064.33805 1.00 3.125 82252 1230 0.95 81125 240 0.96 80134.33 350 0.96 1.562583668 949 0.97 82370 308 0.98 81368.33 812 0.98 0.78125 84072 1686 0.9782812 745 0.98 82135.67 1203 0.99 0.390625 84087 1748 0.97 82723 17520.98 82945.67 1154 1.00 0.1953125 85561 2108 0.99 83059 1024 0.9883526.33 832 1.00 0 86300 1882 1.00 84426 1289 1.00 83352.67 1781 1.00

Example 96 Thienopyrrole Compounds Enable Multiplexing

The following examples describe the use of the thienopyrrole compoundsof the present invention to allow multiplexing of assays which utilizingNluc luciferase and another luciferase, e.g., firefly luciferase.

In a Corning 3570 assay plate, 1× REAL-TIME-GLO™ MT Cell Viability assayreagent was prepared in 40 μL DMEM media (n=3) and incubated overnight.Next, 40 μL of a titration of JRW-0147 or JRW-0344 (2× concentration inCASPASE-GLO® 3/7 Assay Reagent (Promega Corporation)) was added. Thereactions were incubated at room temperature, and luminescencedetermined at 1 hour.

Table 4 shows that the compounds inhibit the background luminescencefrom REALTIME-GLO™ assay in the multiplex with CASPASE-GLO® assay (mediacontaining REALTIME-GLO™ reagent). The compounds inhibit the Nluc enzymeand decrease the background luminescence from REALTIME-GLO™ reagent.

TABLE 4 1 hour [cmpd], JRW-0147 JRW-0344 final μM Avg SD S/B Avg SD S/B100 6568 1159 0.05 5523 956 0.03 50 8136 700 0.06 6790 802 0.04 25 106991130 0.08 9761 674 0.06 12.5 17293 2129 0.12 15547 3856 0.10 6.25 278173812 0.20 19560 3555 0.12 3.125 45167 579 0.32 33523 3740 0.21 1.562565173 1320 0.47 65097 6521 0.41 0.78125 106867 8866 0.77 95993 7772 0.610.390625 117833 5829 0.85 138900 31425 0.88 0.1953125 124500 7192 0.89126333 20215 0.80 0.09765625 117067 8214 0.84 112533 14372 0.71 0 13936711254 1.00 158067 13180 1.00

Example 97 Cell Permeability

The following example demonstrates the permeability of the thienopyrrolecompounds described herein. HEK293 were transiently transfected withBeta-2 Adrenergic Receptor-Nluc (B2AR-Nluc) fusion protein to anchor andorient Nluc cytosolically and pGEM-3z carrier DNA (1:100). 24 hrspost-transfection, cells were treated with −/+50 μg/mL digitonin toemulate a live and lytic scenario, respectively. Then, intact cells andpermeabilized cells were exposed to a compound response curve ofthienopyrrole compound up to 2 hrs. 10 μM furimazine (Promega Corp.) wasadded, and luminescence was measured. If a thienopyrrole compound(inhibitor) was permeable, the dose-response curves for both live andpermeabilized cells overlapped. However, if a thienopyrrole compound wasimpermeable, the EC50 was right-shifted in the live cells relative tothe permeabilized cells. FIGS. 17A-17C demonstrates that JRW-0147 andJRW-0344 are cell impermeable characterized by right shifted EC50 inLive vs. Lytic cells. JRW-0013 served as a cell permeable controlcharacterized by similar EC50 in Live vs Lytic cells.

Example 98 Cell Viability and Toxicity

The following example demonstrates the cell viability and toxicity ofJRW-0344. HEK293 cells were plated at 20 k cells/well and exposed tovehicle (DMSO), digitonin (positive control for cell death), or JRW-0344for 10 min/30 min/120 min/240 min/or 18 hrs. CellTiter-Glo (PromegaCorp.) was added, and luminescence measured. FIGS. 18A-18C demonstratethat there was no apparent toxicity of vehicle or JRW-0344 up to 100 μMafter 18 hrs. Digitonin treated cells experienced almost complete deathat concentrations greater than 2 μg/mL.

Example 99 Cell Permeability with Bioluminescent Imaging

HeLa cells were transiently transfected with Nluc-ADRB2 (extracellularNluc) or ADRB2-Nluc (intracellular Nluc) to anchor and orient Nlucextracellularly or intracellularly, respectively. 24 hrspost-transfection, cells were treated with −/+30 μM of a thienopyrrolecompound, JRW-0147, JRW-0013, and JRW-0344. 10 μM furimazine was added,and luminescence was detected by imaging on the Olympus LV200. DMSO(untreated) was used as a negative control and did not inhibit Nluc ineither orientation. JRW-0013 is a cell permeable positive control andinhibited Nluc in either orientation. However, when Nluc is orientatedextracellularly, JRW-0344 or JRW-0147 inhibit the classic ring-likestructure, but not when Nluc is orientated intracellularly. See FIG. 19.

Example 100 Inhibition of Extracellular Luciferase Activity

The following example demonstrates the ability of the thienopyrrolecompounds described herein to inhibit extracellular luciferase activitywhile enhancing intracellular BRET. See FIG. 20A. HEK293 weretransiently transfected with either SRC-Nluc or Nluc-HaloTag. 24 hrspost-transfection, 20 k/cells were plated into wells of a 96-well plate(Costar 3600). 1 μM of the cell impermeable tracer Dasatinib-DY605 wasadded to the HEK293 SRC-Nluc expressing cells, and 100 nM of the cellpermeable tracer NanoBRET-618, was added to the Nluc-HaloTag expressingcells. Both HEK293 SRC-Nluc and Nluc-HaloTag cells were exposed to adose-response curve of JRW-0344. FIGS. 20A-20C demonstrate that JRW-0344inhibits BRET in cell debris in the HEK293 SRC-Nluc expressing cells,but not in HEK293 Nluc-HaloTag expressing cells.

Example 101 Inhibitor IC50 Determination

The following example provides the IC50 values for the compoundsdisclosed herein. See Table 5 NANOLUC® enzyme was diluted to 0.4 ng/mlin CO2 independent media+10% FBS to make the detection reagent. A 3×dilution series of each inhibitor was then made in the detectionreagent. A “no inhibitor” control was also made for each sample. 50 ulof each inhibitor dilution was mixed with 50 ul of NanoGlo buffercontaining 20 uM furimazine. (Final furimazine concentration is 10 uMwhich is at Km.), and luminescence measured. Each sample was normalizedto the “no inhibitor” control. The IC50 values were then determinedusing GraphPad Prism (log[inhibitor] vs. normalized response).

TABLE 5 Inhibitor IC50 (uM) jrw-0004 1.1 wz-141-74 16.4 wz-141-84 5.5wz-141-88 0.54 jrw-0013 0.14 jrw-0009 2.7 jrw-0008 1.1 jrw-0006 0.26jrw-0034 0.059 jrw-0041 0.2 jrw-0042 0.31 jrw-0043 0.84 jrw-0044 16.8jrw-0051 0.74 jrw-0052 1.3 jrw-0081 19.4 jrw-0109 10.1 jrw-0110 3jrw-0138 0.13 jrw-0143 2.8 jrw-0143 2.6 jrw-0145 0.12 jrw-0147 0.094jrw-0148 5.3 jrw-0149 2.5 jrw-0151 7 jrw-0152 4.3 jrw-0188 0.022jrw-0195 0.48 jrw-0198 1 jrw-0200 0.078 jrw-0208 0.82 jrw-0241 0.063jrw-0242 0.035 jrw-0243 0.21 jrw-0251 0.077 jrw-0344 0.077 jrw-0264 0.24jrw-0268 4.3 jrw-0318 0.11 jrw-0321 0.16 jrw-0322 0.2 jrw-0326 0.0039jrw-0327 0.11 jrw-0330 0.7 jrw-0331 16.9 jrw-0334 1.3 jrw-0335 1.9jrw-0355 0.052 jrw-0359 3 jrw-0360 0.61 jrw-0424 0.005 jrw-0429 0.0021jrw-0430 0.0021 jrw-0432 0.0031 jrw-0434 0.0019 jrw-0456 7.4 jrw-04600.12 jrw-0461 0.077 jrw-0463 0.71 jrw-0466 0.0018 jrw-0478 0.065jrw-0508 0.027

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

For reasons of completeness, various aspects of the present disclosureare set out in the following numbered clauses:

Clause 1. A compound of formula (I), or a salt thereof:

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl; and

R³ and R⁴ are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring.

Clause 2. The compound of clause 1, wherein n is 1.

Clause 3. The compound of any of clauses 1-2, wherein the dashed linerepresents the presence of a bond, and X is CH.

Clause 4. The compound of any of clauses 1-3, wherein A is a 5-memberedheteroaryl ring.

Clause 5. The compound of any of clauses 1-4, wherein A is a thienylring or a furanyl ring.

Clause 6. The compound of any of clauses 1-4, wherein A is a phenylring.

Clause 7. The compound of any of clauses 1-6, wherein R¹ is selectedfrom the group consisting of hydrogen, C₁-C₈ alkyl, halo-C₁-C₈-alkyl,alkoxyalkoxyalkyl and arylalkyl.

Clause 8. The compound of any of clauses 1-7, wherein R¹ is selectedfrom the group consisting of hydrogen, ethyl, n-hexyl,2-(2-methoxyethoxy)ethyl and benzyl.

Clause 9. The compound of any of clauses 1-8, wherein R¹ is ethyl.

Clause 10. The compound of any of clauses 1-9, wherein R² is optionallysubstituted aryl.

Clause 11. The compound of any of clauses 1-10, wherein R² issubstituted phenyl.

Clause 12. The compound of clause 11, wherein R² is phenyl substitutedwith one substituent selected from the group consisting of C₁-C₄ alkyl,cyano, amido, C₁-C₄ alkoxy, and hydroxyalkyl.

Clause 13. The compound of any of clauses 1-12, wherein R² is phenylsubstituted with one methyl group.

Clause 14. The compound of any of clauses 1-13, wherein R³ and R⁴,together with the nitrogen atom to which they are attached, togetherform an optionally substituted ring.

Clause 15. The compound of any of clauses 1-14, wherein R³ and R⁴,together with the nitrogen atom to which they are attached, togetherform an optionally substituted monocyclic heterocycle.

Clause 16. The compound of any of clauses 1-15, wherein the optionallysubstituted monocyclic heterocycle is selected from the group consistingof optionally substituted pyrrolidine, piperidine and piperazine.

Clause 17. The compound of any of clauses 1-16, wherein the optionallysubstituted monocyclic heterocycle is selected from the group consistingof unsubstituted pyrrolidine, unsubstituted piperidine, piperidinesubstituted with one substituent, or piperazine substituted with onesubstituent.

Clause 18. The compound of any of clauses 1-17, wherein R³ is hydrogen.

Clause 19. The compound of any of clauses 1-18, wherein R⁴ is selectedfrom the group consisting of unsubstituted C₁-C₈ alkyl,carboxy-C₁-C₈-alkyl, C₁-C₄-alkoxycarbonyl-C₁-C₈-alkyl, optionallysubstituted phenyl, optionally substituted C₅-C₆ cycloalkyl, optionallysubstituted heterocyclyl, optionally substituted heteroarylalkyl, andoptionally substituted heterocyclylalkyl.

Clause 20. The compound of any of clauses 1-19, wherein R⁴ is phenylthat is unsubstituted or substituted with one substituent.

Clause 21. The compound of clause 20, wherein the substituent isselected from the group consisting of C₁-C₄-alkoxycarbonyl andC₁-C₄-alkoxycarbonyl-C₁-C₄-alkyl.

Clause 22. The compound of any of clauses 1-18, wherein R⁴ is cyclohexylsubstituted with one substituent selected from the group consisting ofcarboxy, C₁-C₄-alkoxycarbonyl, C₁-C₈-alkylamido,hydroxy-C₁-C₈-alkylamido, amido, optionally substitutedamino-C₁-C₈-alkylamido, C₁-C₄-dialkylamino-C₁-C₈-alkylamido,carboxy-C₁-C₈-alkylamido, sulfonic acid-C₁-C₈-alkylamido,sulfonate-C₁-C₈-alkylamido, C₁-C₄-alkylcarbonyl-C₁-C₈-alkylamido,optionally substituted C₃-C₆-cycloalkylamido, and optionally substitutedheterocyclylamido.

Clause 23. The compound of any of clauses 1-22, wherein the compound hasformula (Ia):

Clause 24. The compound of any of clauses 1-23, wherein the compound hasformula (Ib):

wherein:

Y is selected from the group consisting of —NR^(a)R^(b) and —OR^(c);

R^(a) and R^(b) are each independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₈ alkyl, optionallysubstituted C₃-C₈-cycloalkyl, and optionally substituted heterocyclyl;or R^(a) and R^(b), together with the nitrogen atom to which they areattached, together form an optionally substituted ring; and

R^(c) is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl.

Clause 25. The compound of clause 24, wherein Y is —OR^(c).

Clause 26. The compound of any of clauses 24-25, wherein R^(c) isselected from the group consisting of hydrogen and methyl.

Clause 27. The compound of clause 24, wherein Y is —NR^(a)R^(b).

Clause 28. The compound of clause 27, wherein R^(a) and R^(b), togetherwith the nitrogen atom to which they are attached, together form anoptionally substituted ring.

Clause 29. The compound of any of clauses 27-28, wherein R^(a) andR^(b), together with the nitrogen atom to which they are attached,together form an optionally substituted monocyclic heterocycle.

Clause 30. The compound of clause 29, wherein the optionally substitutedmonocyclic heterocycle is optionally substituted piperidine.

Clause 31. The compound of any of clauses 29-30, wherein the optionallysubstituted monocyclic heterocycle is selected from the group consistingof unsubstituted piperidine and piperidine substituted with onesubstituent.

Clause 32. The compound of clause 27, wherein R^(a) is hydrogen.

Clause 33. The compound of clause 32, wherein R^(b) is selected from thegroup consisting of hydrogen, optionally substituted C₁-C₈ alkyl,optionally substituted C₃-C₈-cycloalkyl, and optionally substitutedheterocyclyl.

Clause 34. The compound of any of clauses 31-32, wherein R^(b) isselected from the group consisting of hydrogen, C₁-C₆ alkyl,hydroxyalkyl, optionally substituted aminoalkyl, carboxyalkyl, sulfonicacid-alkyl, sulfonate-alkyl, alkylcarbonylalkyl, optionally substitutedC₃-C₆-cycloalkyl, and optionally substituted six-membered heterocyclyl.

Clause 35. The compound of any of clauses 1-34, wherein the compound hasthe following formula (Ib′)

Clause 36. The compound of any of clauses 1-35, wherein the compound isselected from the group consisting of:

N-cyclohexyl-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-ethyl-2-(5-(pyrrolidine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N4m-tolyl)acetamide;

N-ethyl-2-(5-(piperidine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide;

ethyl1-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)piperidine-4-carboxylate;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-phenyl-4H-thieno[3,2-b]pyrrole-5-carboxamide;

ethyl2-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)phenyl)acetate;

methyl3-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)benzoate;

methyl-cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

8-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)octanoicacid;

6-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)piperidin-1-yl)-6-oxohexanoicacid;

trans-methyl-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid;

N-(trans-4-(butylcarbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((2-hydroxyethyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-(trans-4-((2-(dimethylamino)ethyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)butanoicacid;

N-(trans-4-carbamoylcyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-(hexylcarbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

ethyl1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carbonyl)piperidine-4-carboxylate;

methyl6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoate;

6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoicacid;

1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carbonyl)piperidine-4-carboxylicacid;

8-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)octanoicacid;

N-(trans-4-(cyclohexylcarbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((1-methylpiperidin-4-yl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

tert-butyl4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)piperidine-1-carboxylate;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-(piperidin-4-ylcarbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-(trans-4-((1-acetylpiperidin-4-yl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

tert-butyl(6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexyl)carbamate;

N-(trans-4-((6-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5(6)-carboxamido)hexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-(trans-4-((6-aminohexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamidehydrochloride;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

methyl-trans-4-(trans-4-(4-(2-(ethcyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)cyclohexane-1-carboxylate;

trans-4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)cyclohexane-1-carboxylicacid;

(11S,14S,17S)-17-acetamido-11,14-bis(carboxymethyl)-1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexyl)-1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecan-19-oicacid;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-methyl-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-cyclopentyl-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(pyridin-4-ylmethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(3-morpholinopropyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-ethyl-2-(5-(4-methylpiperazine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide;

methyl4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate;

N-cyclohexyl-4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-cyclohexyl-4-(24(2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

methyl-trans-4-(4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyl4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate;

N-cyclohexyl-4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

methyl-trans-4-(4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyl4-(2-(benzyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate;

6-(cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoicacid;

methyl6-(trans-4-((4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)methyl)cyclohexane-1-carboxamido)hexanoate;

6-(trans-4-((4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)methyl)cyclohexane-1-carboxamido)hexanoicacid;

sodium6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;

potassium6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;

trans-4-(4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid;

methyltrans-4-(4-(2-(ethyl(phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-((3-cyanophenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-((3-carbamoylphenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(3-methoxyphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(o-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(6-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(p-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6-methoxy-1H-indole-2-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-furo[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;

sodium6-(trans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;

methyltrans-4-(4-(2-(ethyl(3-isopropylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(3-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-((3-(bromomethyl)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-((3-(dimethylamino)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(4-(2-(ethyl(3-isobutylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;

methyltrans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate;

1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-1H-indole-2-carboxamide;

sodium6-(trans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;

methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-pyrrole-2-carboxamido)cyclohexane-1-carboxylate;and

methyltrans-4-(6-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6H-thieno[2,3-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate.

Clause 37. A method of inhibiting an Oplophorus-derived luciferase themethod comprising contacting the Oplophorus-derived luciferase with acompound of any of clauses 1-36.

Clause 38. The method of clause 37, wherein the Oplophorus-derivedluciferase comprises a polypeptide sequence of SEQ ID NO: 2.

Clause 39. A method of inhibiting an Oplophorus-derived luciferase, themethod comprising contacting the Oplophorus-derived luciferase with acompound of formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring.

Clause 40. The method of clause 39, wherein the Oplophorus-derivedluciferase comprises a polypeptide sequence of SEQ ID NO: 2.

Clause 41. A method for modulating luminescence of an Oplophorus-derivedluciferase in a sample, the method comprising,

(a) contacting the sample with a coelenterazine substrate and thecompound of any of clauses 1-36; and

(b) detecting luminescence in the sample,

wherein the compound of any of clauses 1-36 causes a decrease in theluminescence from the Oplophorus-derived luciferase.

Clause 42. A method to detect an interaction between a first protein anda second protein in a sample, the method comprising:

(a) contacting a sample with a coelenterazine substrate and the compoundof any one of clauses 1-36, wherein the sample comprises:

-   -   (ix) a first polynucleotide encoding a first fusion protein,        wherein the first fusion protein comprises a first fragment of        an Oplophorus-derived luciferase and a first protein; and    -   (x) a second polynucleotide encoding a second fusion protein,        wherein the second fusion protein comprises a second fragment of        the Oplophorus-derived luciferase and a second protein; and

(b) detecting luminescence in the sample,

wherein the detection of luminescence indicates an interaction betweenthe first protein and the second protein.

Clause 43. The method of any of clauses 41-42, comprising contacting thesample with the coelenterazine substrate prior to contacting the samplewith the compound of any one of clauses 1-36.

Clause 44. The method of clause 42, wherein when the first protein andsecond protein interact, the first fragment of the Oplophorus-derivedluciferase and the second fragment of the Oplophorus-derived luciferasereconstitute a full-length enzyme capable of stably binding thecoelenterazine substrate.

Clause 45. A method to detect an interaction between a first protein anda second protein in a sample, the method comprising:

(a) contacting a sample with a coelenterazine substrate and the compoundof any one of clauses 1-36, wherein the sample comprises:

-   -   (xi) a first polynucleotide encoding a first fusion protein,        wherein the first fusion protein comprises an Oplophorus-derived        luciferase and a first protein, wherein the Oplophorus-derived        luciferase is a bioluminescent donor; and    -   (xii) a second polynucleotide encoding a second fusion protein,        wherein the second fusion protein comprises a fluorescent        acceptor molecule and a second protein;

(b) detecting bioluminescence resonance energy transfer (BRET) in thesample, indicating an interaction or close proximity of thebioluminescent donor and and the fluorescence acceptor.

Clause 46. The method of any one of clauses 41-45, wherein the samplecomprises a cell.

Clause 47. The method of clause 46, wherein the cell comprises theOplophorus-derived luciferase.

Clause 48. The method of clause 46, wherein the cell expresses theOplophorus-derived luciferase.

Clause 49. The method of any one of clauses 41-48, wherein thecoelenterazine substrate is a coelenterazine, coelenterazinederivatives, coelenterazine analogs, pro-coelenterazine, orquinone-masked coelenterazine.

Clause 50. A bioluminescence resonance energy transfer (BRET) systemcomprising: a first fusion protein including a first target protein anda bioluminescence donor molecule, wherein the bioluminescence donormolecule is an Oplophorus-derived luciferase ; a second fusion proteinincluding a second target protein and a fluorescent acceptor molecule; acoelenterazine substrate, and the compound of any one of clauses 1-36.

Clause 51. A kit comprising:

(a) a compound of any of clauses 1-36; and

(b) an Oplophorus-derived luciferase.

Clause 52. The kit of clause 51, wherein the Oplophorus-derivedluciferase comprises a polypeptide sequence of SEQ ID NO: 2

Clause 53. The kit of any of clauses 51-52, further comprising acoelenterazine substrate.

Clause 54. The kit of any of clauses 51-53, further comprisinginstructions for carrying out a luminescent assay.

Clause 55. A method for modulating luminescence of an Oplophorus-derivedluciferase in a sample, the method comprising:

(a) contacting the sample with a coelenterazine substrate and a compoundof formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring; and

(b) detecting luminescence in the sample,

wherein the compound of formula (II) causes a decrease in theluminescence from the Oplophorus-derived luciferase.

Clause 56. A method to detect an interaction between a first protein anda second protein in a sample, the method comprising:

(a) contacting a sample with a coelenterazine substrate and a compoundof formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring,

wherein the sample comprises:

-   -   (xiii) a first polynucleotide encoding a first fusion protein,        wherein the first fusion protein comprises a first fragment of        an Oplophorus-derived luciferase and a first protein; and    -   (xiv) a second polynucleotide encoding a second fusion protein,        wherein the second fusion protein comprises a second fragment of        the Oplophorus-derived luciferase and a second protein; and

(b) detecting luminescence in the sample,

wherein the detection of luminescence indicates an interaction betweenthe first protein and the second protein.

Clause 57. The method of any of clauses 55-56, comprising contacting thesample with the coelenterazine substrate prior to contacting the samplewith the compound of formula (II).

Clause 58. The method of clause 57, wherein when the first protein andsecond protein interact, the first fragment of the Oplophorus-derivedluciferase and the second fragment of the Oplophorus-derived luciferasereconstitute a full-length enzyme capable of stably binding thecoelenterazine substrate.

Clause 59. A method to detect an interaction between a first protein anda second protein in a sample, the method comprising:

(a) contacting a sample with a coelenterazine substrate and a compoundof formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring,

wherein the sample comprises:

-   -   (xv) a first polynucleotide encoding a first fusion protein,        wherein the first fusion protein comprises an Oplophorus-derived        luciferase and a first protein, wherein the Oplophorus-derived        luciferase is a bioluminescent donor; and    -   (xvi) a second polynucleotide encoding a second fusion protein,        wherein the second fusion protein comprises a fluorescent        acceptor molecule and a second protein;

(b) detecting bioluminescence resonance energy transfer (BRET) in thesample, indicating an interaction or close proximity of thebioluminescent donor and the fluorescence acceptor.

Clause 60. The method of any one of clauses 55-59, wherein the samplecomprises a cell.

Clause 61. The method of clause 60, wherein the cell comprises theOplophorus-derived luciferase.

Clause 62. The method of clause 60, wherein the cell expresses theOplophorus-derived luciferase.

Clause 63. The method of any one of clauses 54-62, wherein thecoelenterazine substrate is coelenterazine substrate is acoelenterazine, coelenterazine derivatives, coelenterazine analogs,pro-coelenterazine, or quinone-masked coelenterazine.

Clause 64. A bioluminescence resonance energy transfer (BRET) systemcomprising: a first fusion protein including a first target protein anda bioluminescence donor molecule, wherein the bioluminescence donormolecule is an Oplophorus-derived luciferase ; a second fusion proteinincluding a second target protein and a fluorescent acceptor molecule; acoelenterazine substrate, and a compound of formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkylyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring.

Clause 65. A kit comprising:

(a) a compound of formula (II):

wherein:

the dashed line represents the presence or absence of a bond;

n is 0, 1, 2, 3, 4 or 5;

X is CH, N, O, or S;

-   -   wherein, when the dashed line represents the presence of a bond,        X is CH or N, and when the dashed line represents the absence of        a bond, X is O or S;

A is an optionally substituted phenyl ring, or an optionally substituted5- or 6-membered heteroaryl ring;

R¹ and R² are each independently selected from the group consisting ofhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl, optionally substituted alkoxyalkyl and optionally substitutedalkoxyalkoxyalkyl;

Z is selected from the group consisting of —NR³R⁴ and —OR⁵; and

R³, R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈-cycloalkyl, optionally substituted C₃-C₈-cycloalkylalkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, optionally substitutedheteroarylalkyl, optionally substituted heterocyclyl, and optionallysubstituted heterocyclylalkyl; or R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring; and

(b) an Oplophorus-derived luciferase.

Clause 66. The kit of clause 65, wherein the Oplophorus-derivedluciferase comprises a polypeptide sequence of SEQ ID NO: 2.

Clause 67. The kit of any of clauses 65-66, further comprising acoelenterazine substrate.

Clause 68. The kit of any of clauses 65-67, further comprisinginstructions for carrying out a luminescent assay.

APPENDIX SEQ ID NO: 1 - Native Mature Oplophorus luciferaseamino acid sequence FTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPDGSLLFR- VTIN GVTGWRLCENILA SEQ ID NO: 2 - Nluc amino acid sequenceMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGS LLFRVTINGVTGWRLCERILA

1. A compound of formula (I), or a salt thereof:

wherein: the dashed line represents the presence or absence of a bond; nis 0, 1, 2, 3, 4 or 5; X is CH, N, O, or S; wherein, when the dashedline represents the presence of a bond, X is CH or N, and when thedashed line represents the absence of a bond, X is O or S; A is anoptionally substituted phenyl ring, or an optionally substituted 5- or6-membered heteroaryl ring; R¹ and R² are each independently selectedfrom the group consisting of hydrogen, optionally substituted C₁-C₈alkyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, optionally substituted alkoxyalkyl andoptionally substituted alkoxyalkoxyalkyl; and R³ and R⁴ are eachindependently selected from the group consisting of hydrogen, optionallysubstituted C₁-C₈ alkyl, optionally substituted C₃-C₈-cycloalkyl,optionally substituted C₃-C₈-cycloalkylalkyl, optionally substitutedaryl, optionally substituted arylalkyl, optionally substitutedheteroaryl, optionally substituted heteroarylalkyl, optionallysubstituted heterocyclyl, and optionally substituted heterocyclylalkyl;or R³ and R⁴, together with the nitrogen atom to which they areattached, together form an optionally substituted ring.
 2. The compoundof claim 1, wherein n is
 1. 3. The compound of any of claims 1-2,wherein the dashed line represents the presence of a bond, and X is CH.4. The compound of any of claims 1-3, wherein A is a 5-memberedheteroaryl ring.
 5. The compound of any of claims 1-4, wherein A is athienyl ring or a furanyl ring.
 6. The compound of any of claims 1-3,wherein A is a phenyl ring.
 7. The compound of any of claims 1-6,wherein R¹ is selected from the group consisting of hydrogen, C₁-C₈alkyl, halo-C₁-C₈-alkyl, alkoxyalkoxyalkyl and arylalkyl.
 8. Thecompound of any of claims 1-7, wherein R¹ is selected from the groupconsisting of hydrogen, ethyl, n-hexyl, 2-(2-methoxyethoxy)ethyl andbenzyl.
 9. The compound of any of claims 1-8, wherein R¹ is ethyl. 10.The compound of any of claims 1-9, wherein R² is optionally substitutedaryl.
 11. The compound of claim 10, wherein R² is substituted phenyl.12. The compound of claim 11, wherein R² is phenyl substituted with onesubstituent selected from the group consisting of C₁-C₄ alkyl, cyano,amido, C₁-C₄ alkoxy, and hydroxyalkyl.
 13. The compound of claim 12,wherein R² is phenyl substituted with one methyl group.
 14. The compoundof any of claims 1-13, wherein R³ and R⁴, together with the nitrogenatom to which they are attached, together form an optionally substitutedring.
 15. The compound of claim 14, wherein R³ and R⁴, together with thenitrogen atom to which they are attached, together form an optionallysubstituted monocyclic heterocycle.
 16. The compound of claim 15,wherein the optionally substituted monocyclic heterocycle is selectedfrom the group consisting of optionally substituted pyrrolidine,piperidine and piperazine.
 17. The compound of claim 15, wherein theoptionally substituted monocyclic heterocycle is selected from the groupconsisting of unsubstituted pyrrolidine, unsubstituted piperidine,piperidine substituted with one substituent, or piperazine substitutedwith one substituent.
 18. The compound of any of claims 1-17, wherein R³is hydrogen.
 19. The compound of any of claims 1-18, wherein R⁴ isselected from the group consisting of unsubstituted C₁-C₈ alkyl,halo-C₁-C₈-alkyl, carboxy-C₁-C₈-alkyl, C₁-C₄-alkoxycarbonyl-C₁-C₈-alkyl,optionally substituted phenyl, optionally substituted C₅-C₆ cycloalkyl,optionally substituted C₅-C₆-cycloalkylalkyl, optionally substitutedheterocyclyl, optionally substituted heteroarylalkyl, and optionallysubstituted heterocyclylalkyl.
 20. The compound of claim 19, wherein R⁴is phenyl that is unsubstituted or substituted with one substituent. 21.The compound of claim 20, wherein the substituent is selected from thegroup consisting of C₁-C₄-alkoxycarbonyl andC₁-C₄-alkoxycarbonyl-C₁-C₄-alkyl.
 22. The compound of any of claims1-18, wherein R⁴ is cyclohexyl substituted with one substituent selectedfrom the group consisting of carboxy, C₁-C₄-alkoxycarbonyl,C₁-C₈-alkylamido, hydroxy-C₁-C₈-alkylamido, amido, optionallysubstituted amino-C₁-C₈-alkylamido, C₁-C₄-dialkylamino-C₁-C₈-alkylamido,carboxy-C₁-C₈-alkylamido, sulfonic acid-C₁-C₈-alkylamido,sulfonate-C₁-C₈-alkylamido, C₁-C₄-alkylcarbonyl-C₁-C₈-alkylamido,optionally substituted C₃-C₆-cycloalkylamido, and optionally substitutedheterocyclylamido.
 23. The compound of claim 1, wherein the compound hasformula (Ia):


24. The compound of claim 1, wherein the compound has formula (Ib):

wherein: Y is selected from the group consisting of —NR^(a)R^(b) and—OR^(c); R^(a) and R^(b) are each independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₈ alkyl, optionallysubstituted C₃-C₈-cycloalkyl, and optionally substituted heterocyclyl;or R^(a) and R^(b), together with the nitrogen atom to which they areattached, together form an optionally substituted ring; and R^(c) isselected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl.
 25. The compound of claim 24, wherein Y is—OR^(c).
 26. The compound of claim 25, wherein R^(c) is selected fromthe group consisting of hydrogen and methyl.
 27. The compound of claim24, wherein Y is —NR^(a)R^(b).
 28. The compound of claim 27, whereinR^(a) and R^(b), together with the nitrogen atom to which they areattached, together form an optionally substituted ring.
 29. The compoundof claim 28, wherein R^(a) and R^(b), together with the nitrogen atom towhich they are attached, together form an optionally substitutedmonocyclic heterocycle.
 30. The compound of claim 29, wherein theoptionally substituted monocyclic heterocycle is optionally substitutedpiperidine.
 31. The compound of claim 30, wherein the optionallysubstituted monocyclic heterocycle is selected from the group consistingof unsubstituted piperidine and piperidine substituted with onesubstituent.
 32. The compound of claim 27, wherein R^(a) is hydrogen.33. The compound of claim 32, wherein R^(b) is selected from the groupconsisting of hydrogen, optionally substituted C₁-C₈ alkyl, optionallysubstituted C₃-C₈-cycloalkyl, and optionally substituted heterocyclyl.34. The compound of claim 32, wherein R^(b) is selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, hydroxyalkyl, optionallysubstituted aminoalkyl, carboxyalkyl, sulfonic acid-alkyl,sulfonate-alkyl, alkylcarbonylalkyl, optionally substitutedC₃-C₆-cycloalkyl, and optionally substituted six-membered heterocyclyl.35. The compound of any of claims 24-34, wherein the compound has thefollowing formula (Ib′)


36. The compound of claim 1, wherein the compound is selected from thegroup consisting of:N-cyclohexyl-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;N-ethyl-2-(5-(pyrrolidine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide;N-ethyl-2-(5-(piperidine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide;ethyl1-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)piperidine-4-carboxylate;4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-phenyl-4H-thieno[3,2-b]pyrrole-5-carboxamide;ethyl2-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)phenyl)acetate;methyl3-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)benzoate;methyl-cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;8-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)octanoicacid;6-(4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)piperidin-1-yl)-6-oxohexanoicacid;trans-methyl-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid;N-(trans-4-(butylcarbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((2-hydroxyethyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;N-(trans-4-((2-(dimethylamino)ethyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)butanoicacid;N-(trans-4-carbamoylcyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-(hexylcarbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;ethyl1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carbonyl)piperidine-4-carboxylate;methyl6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoate;6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoicacid;1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carbonyl)piperidine-4-carboxylicacid;8-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)octanoicacid;N-(trans-4-(cyclohexylcarbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((1-methylpiperidin-4-yl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;tert-butyl4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)piperidine-1-carboxylate;4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-(piperidin-4-ylcarbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;N-(trans-4-((1-acetylpiperidin-4-yl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;tert-butyl(6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexyl)carbamate;N-(trans-4-((6-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5(6)-carboxamido)hexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;N-(trans-4-((6-aminohexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamidehydrochloride;4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;methyl-trans-4-(trans-4-(4-(2-(ethcyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)cyclohexane-1-carboxylate;trans-4-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)cyclohexane-1-carboxylicacid;(11S,14S,17S)-17-acetamido-11,14-bis(carboxymethyl)-1-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexyl)-1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecan-19-oicacid;4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-methyl-4H-thieno[3,2-b]pyrrole-5-carboxamide;N-cyclopentyl-4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(pyridin-4-ylmethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-N-(3-morpholinopropyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;N-ethyl-2-(5-(4-methylpiperazine-1-carbonyl)-4H-thieno[3,2-b]pyrrol-4-yl)-N-(m-tolyl)acetamide;methyl4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate;N-cyclohexyl-4-(2-oxo-2-(m-tolylamino)ethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;N-cyclohexyl-4-(24(2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;methyl-trans-4-(4-(2-((2-(2-methoxyethoxy)ethyl)(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyl4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate;N-cyclohexyl-4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;methyl-trans-4-(4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyl4-(2-(benzyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate;6-(cis-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexanoicacid; methyl6-(trans-4-((4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)methyl)cyclohexane-1-carboxamido)hexanoate;6-(trans-4-((4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)methyl)cyclohexane-1-carboxamido)hexanoicacid; sodium6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;potassium6-(trans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;trans-4-(4-(2-(hexyl(m-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylicacid; methyltrans-4-(4-(2-(ethyl(phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-((3-cyanophenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-((3-carbamoylphenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-(ethyl(3-methoxyphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-(ethyl(o-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-(6-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-(ethyl(p-tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-(ethyl(4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6-methoxy-1H-indole-2-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-4H-furo[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide;sodium6-(trans-4-(4-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;methyltrans-4-(4-(2-(ethyl(3-isopropylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-(ethyl(3-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-((3-(bromomethyl)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-((3-(dimethylamino)phenyl)(ethyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(4-(2-(ethyl(3-isobutylphenyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate;methyltrans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxylate;1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-N-(trans-4-((6-hydroxyhexyl)carbamoyl)cyclohexyl)-1H-indole-2-carboxamide;sodium6-(trans-4-(1-(2-(ethyl(3-ethylphenyl)amino)-2-oxoethyl)-1H-indole-2-carboxamido)cyclohexane-1-carboxamido)hexane-1-sulfonate;methyltrans-4-(1-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-1H-pyrrole-2-carboxamido)cyclohexane-1-carboxylate;and methyltrans-4-(6-(2-(ethyl(m-tolyl)amino)-2-oxoethyl)-6H-thieno[2,3-b]pyrrole-5-carboxamido)cyclohexane-1-carboxylate.37. A method of inhibiting an Oplophorus-derived luciferase the methodcomprising contacting the Oplophorus-derived luciferase with a compoundof any of claims 1-36.
 38. The method of claim 37, wherein theOplophorus-derived luciferase comprises a polypeptide sequence of SEQ IDNO:
 2. 39. A method of inhibiting an Oplophorus-derived luciferase, themethod comprising contacting the Oplophorus-derived luciferase with acompound of formula (II):

wherein: the dashed line represents the presence or absence of a bond; nis 0, 1, 2, 3, 4 or 5; X is CH, N, O, or S; wherein, when the dashedline represents the presence of a bond, X is CH or N, and when thedashed line represents the absence of a bond, X is O or S; A is anoptionally substituted phenyl ring, or an optionally substituted 5- or6-membered heteroaryl ring; R¹ and R² are each independently selectedfrom the group consisting of hydrogen, optionally substituted C₁-C₈alkyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, optionally substituted alkoxyalkyl andoptionally substituted alkoxyalkoxyalkyl; Z is selected from the groupconsisting of —NR³R⁴ and —OR⁵; and R³, R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈-cycloalkyl, optionallysubstituted C₃-C₈-cycloalkylalkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, optionally substitutedheterocyclyl, and optionally substituted heterocyclylalkyl; or R³ andR⁴, together with the nitrogen atom to which they are attached, togetherform an optionally substituted ring.
 40. The method of claim 39, whereinthe Oplophorus-derived luciferase comprises a polypeptide sequence ofSEQ ID NO:
 2. 41. A method for modulating luminescence of anOplophorus-derived luciferase in a sample, the method comprising, (a)contacting the sample with a coelenterazine substrate and the compoundof any of claims 1-36; and (b) detecting luminescence in the sample,wherein the compound of any of claims 1-36 causes a decrease in theluminescence from the Oplophorus-derived luciferase.
 42. A method todetect an interaction between a first protein and a second protein in asample, the method comprising: (a) contacting a sample with acoelenterazine substrate and the compound of any one of claims 1-36,wherein the sample comprises: (xvii) a first polynucleotide encoding afirst fusion protein, wherein the first fusion protein comprises a firstfragment of an Oplophorus-derived luciferase and a first protein; and(xviii) a second polynucleotide encoding a second fusion protein,wherein the second fusion protein comprises a second fragment of theOplophorus-derived luciferase and a second protein; and (b) detectingluminescence in the sample, wherein the detection of luminescenceindicates an interaction between the first protein and the secondprotein.
 43. The method of any of claims 41-42, comprising contactingthe sample with the coelenterazine substrate prior to contacting thesample with the compound of any one of claims 1-36.
 44. The method ofclaim 42, wherein when the first protein and second protein interact,the first fragment of the Oplophorus-derived luciferase and the secondfragment of the Oplophorus-derived luciferase reconstitute a full-lengthenzyme capable of stably binding the coelenterazine substrate.
 45. Amethod to detect an interaction between a first protein and a secondprotein in a sample, the method comprising: (a) contacting a sample witha coelenterazine substrate and the compound of any one of claims 1-36,wherein the sample comprises: (xix) a first polynucleotide encoding afirst fusion protein, wherein the first fusion protein comprises anOplophorus-derived luciferase and a first protein, wherein theOplophorus-derived luciferase is a bioluminescent donor; and (xx) asecond polynucleotide encoding a second fusion protein, wherein thesecond fusion protein comprises a fluorescent acceptor molecule and asecond protein; (b) detecting bioluminescence resonance energy transfer(BRET) in the sample, indicating an interaction or close proximity ofthe bioluminescent donor and the fluorescence acceptor.
 46. The methodof any one of claims 41-45, wherein the sample comprises a cell.
 47. Themethod of claim 46, wherein the cell comprises the Oplophorus-derivedluciferase.
 48. The method of claim 46, wherein the cell expresses theOplophorus-derived luciferase.
 49. The method of any one of claims41-48, wherein the coelenterazine substrate is a coelenterazine,coelenterazine derivatives, coelenterazine analogs, pro-coelenterazine,or quinone-masked coelenterazine.
 50. A bioluminescence resonance energytransfer (BRET) system comprising: a first fusion protein including afirst target protein and a bioluminescence donor molecule, wherein thebioluminescence donor molecule is an Oplophorus-derived luciferase ; asecond fusion protein including a second target protein and afluorescent acceptor molecule; a coelenterazine substrate, and thecompound of any one of claims 1-36.
 51. A kit comprising: (a) a compoundof any one of claims 1-36; and (b) an Oplophorus-derived luciferase. 52.The kit of claim 51, wherein the Oplophorus-derived luciferase comprisesa polypeptide sequence of SEQ ID NO:
 2. 53. The kit of any of claims51-52, further comprising a coelenterazine substrate.
 54. The kit of anyof claims 51-53, further comprising instructions for carrying out aluminescent assay.
 55. A method for modulating luminescence of anOplophorus-derived luciferase in a sample, the method comprising: (a)contacting the sample with a coelenterazine substrate and a compound offormula (II):

wherein: the dashed line represents the presence or absence of a bond; nis 0, 1, 2, 3, 4 or 5; X is CH, N, O, or S; wherein, when the dashedline represents the presence of a bond, X is CH or N, and when thedashed line represents the absence of a bond, X is O or S; A is anoptionally substituted phenyl ring, or an optionally substituted 5- or6-membered heteroaryl ring; R¹ and R² are each independently selectedfrom the group consisting of hydrogen, optionally substituted C₁-C₈alkyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, optionally substituted alkoxyalkyl andoptionally substituted alkoxyalkoxyalkyl; Z is selected from the groupconsisting of —NR³R⁴ and —OR⁵; and R³, R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈-cycloalkyl, optionallysubstituted C₃-C₈-cycloalkylalkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, optionally substitutedheterocyclyl, and optionally substituted heterocyclylalkyl; or R³ andR⁴, together with the nitrogen atom to which they are attached, togetherform an optionally substituted ring; and (b) detecting luminescence inthe sample, wherein the compound of formula (II) causes a decrease inthe luminescence from the Oplophorus-derived luciferase.
 56. A method todetect an interaction between a first protein and a second protein in asample, the method comprising: (a) contacting a sample with acoelenterazine substrate and a compound of formula (II):

wherein: the dashed line represents the presence or absence of a bond; nis 0, 1, 2, 3, 4 or 5; X is CH, N, O, or S; wherein, when the dashedline represents the presence of a bond, X is CH or N, and when thedashed line represents the absence of a bond, X is O or S; A is anoptionally substituted phenyl ring, or an optionally substituted 5- or6-membered heteroaryl ring; R¹ and R² are each independently selectedfrom the group consisting of hydrogen, optionally substituted C₁-C₈alkyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, optionally substituted alkoxyalkyl andoptionally substituted alkoxyalkoxyalkyl; Z is selected from the groupconsisting of —NR³R⁴ and —OR⁵; and R³, R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈-cycloalkyl, optionallysubstituted C₃-C₈-cycloalkylalkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, optionally substitutedheterocyclyl, and optionally substituted heterocyclylalkyl; or R³ andR⁴, together with the nitrogen atom to which they are attached, togetherform an optionally substituted ring, wherein the sample comprises: (xxi)a first polynucleotide encoding a first fusion protein, wherein thefirst fusion protein comprises a first fragment of an Oplophorus-derivedluciferase and a first protein; and (xxii) a second polynucleotideencoding a second fusion protein, wherein the second fusion proteincomprises a second fragment of the Oplophorus-derived luciferase and asecond protein; and (b) detecting luminescence in the sample, whereinthe detection of luminescence indicates an interaction between the firstprotein and the second protein.
 57. The method of any of claims 55-56,comprising contacting the sample with the coelenterazine substrate priorto contacting the sample with the compound of formula (II).
 58. Themethod of claim 57, wherein when the first protein and second proteininteract, the first fragment of the Oplophorus-derived luciferase andthe second fragment of the Oplophorus-derived luciferase reconstitute afull-length enzyme capable of stably binding the coelenterazinesubstrate.
 59. A method to detect an interaction between a first proteinand a second protein in a sample, the method comprising: (a) contactinga sample with a coelenterazine substrate and a compound of formula (II):

wherein: the dashed line represents the presence or absence of a bond; nis 0, 1, 2, 3, 4 or 5; X is CH, N, O, or S; wherein, when the dashedline represents the presence of a bond, X is CH or N, and when thedashed line represents the absence of a bond, X is O or S; A is anoptionally substituted phenyl ring, or an optionally substituted 5- or6-membered heteroaryl ring; R¹ and R² are each independently selectedfrom the group consisting of hydrogen, optionally substituted C₁-C₈alkyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, optionally substituted alkoxyalkyl andoptionally substituted alkoxyalkoxyalkyl; Z is selected from the groupconsisting of —NR³R⁴ and —OR⁵; and R³, R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈-cycloalkyl, optionallysubstituted C₃-C₈-cycloalkylalkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, optionally substitutedheterocyclyl, and optionally substituted heterocyclylalkyl; or R³ andR⁴, together with the nitrogen atom to which they are attached, togetherform an optionally substituted ring, wherein the sample comprises:(xxiii) a first polynucleotide encoding a first fusion protein, whereinthe first fusion protein comprises an Oplophorus-derived luciferase anda first protein, wherein the Oplophorus-derived luciferase is abioluminescent donor; and (xxiv) a second polynucleotide encoding asecond fusion protein, wherein the second fusion protein comprises afluorescent acceptor molecule and a second protein; (b) detectingbioluminescence resonance energy transfer (BRET) in the sample,indicating an interaction or close proximity of the bioluminescent donorand the fluorescence acceptor.
 60. The method of any one of claims55-59, wherein the sample comprises a cell.
 61. The method of claim 60,wherein the cell comprises the Oplophorus-derived luciferase.
 62. Themethod of claim 60, wherein the cell expresses the Oplophorus-derivedluciferase.
 63. The method of any one of claims 55-62, wherein thecoelenterazine substrate is coelenterazine substrate is acoelenterazine, coelenterazine derivatives, coelenterazine analogs,pro-coelenterazine, or quinone-masked coelenterazine.
 64. Abioluminescence resonance energy transfer (BRET) system comprising: afirst fusion protein including a first target protein and abioluminescence donor molecule, wherein the bioluminescence donormolecule is an Oplophorus-derived luciferase; a second fusion proteinincluding a second target protein and a fluorescent acceptor molecule; acoelenterazine substrate; and a compound formula (II):

wherein: the dashed line represents the presence or absence of a bond; nis 0, 1, 2, 3, 4 or 5; X is CH, N, O, or S; wherein, when the dashedline represents the presence of a bond, X is CH or N, and when thedashed line represents the absence of a bond, X is O or S; A is anoptionally substituted phenyl ring, or an optionally substituted 5- or6-membered heteroaryl ring; R¹ and R² are each independently selectedfrom the group consisting of hydrogen, optionally substituted C₁-C₈alkyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, optionally substituted alkoxyalkyl andoptionally substituted alkoxyalkoxyalkyl; Z is selected from the groupconsisting of —NR³R⁴ and —OR⁵; and R³, R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈-cycloalkyl, optionallysubstituted C₃-C₈-cy cl oal kyl al kyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, optionally substitutedheterocyclyl, and optionally substituted heterocyclylalkyl; or R³ andR⁴, together with the nitrogen atom to which they are attached, togetherform an optionally substituted ring.
 65. A kit comprising: (a) acompound of formula (II):

wherein: the dashed line represents the presence or absence of a bond; nis 0, 1, 2, 3, 4 or 5; X is CH, N, O, or S; wherein, when the dashedline represents the presence of a bond, X is CH or N, and when thedashed line represents the absence of a bond, X is O or S; A is anoptionally substituted phenyl ring, or an optionally substituted 5- or6-membered heteroaryl ring; R¹ and R² are each independently selectedfrom the group consisting of hydrogen, optionally substituted C₁-C₈alkyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, optionally substituted alkoxyalkyl andoptionally substituted alkoxyalkoxyalkyl; Z is selected from the groupconsisting of —NR³R⁴ and —OR⁵; and R³, R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈-cycloalkyl, optionallysubstituted C₃-C₈-cycloalkylalkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, optionally substitutedheterocyclyl, and optionally substituted heterocyclylalkyl; or R³ andR⁴, together with the nitrogen atom to which they are attached, togetherform an optionally substituted ring; and (b) an Oplophorus-derivedluciferase.
 66. The kit of claim 65, wherein the Oplophorus-derivedluciferase comprises a polypeptide sequence of SEQ ID NO:
 2. 67. The kitof any of claims 65-66, further comprising a coelenterazine substrate.68. The kit of any of claims 65-67, further comprising instructions forcarrying out a luminescent assay.